Tumor microenvironment response type nano assembly as well as preparation method and application thereof

Abstract

The invention relates to a tumor microenvironment response type nano assembly as well as a preparation method and application thereof, and belongs to the technical fields of pharmaceutical preparation combined treatment, new auxiliary materials and new dosage forms. The invention discloses a tumor microenvironment response type nano assembly which is formed by co-assembling DPA-MOF nanoparticles, manganese dioxide and a porphyrin photosensitizer through intermolecular force and is modified by adopting a PEG (Polyethylene Glycol) modifier. Wherein the mass ratio of the DPA-MOF nanoparticles to the manganese dioxide to the porphyrin photosensitizer is 1: (0.1-10): (0.1-15), and the mass ratio of the sum of the DPA-MOF nanoparticles, the manganese dioxide and the porphyrin photosensitizer to the PEG modifier is 1: (0.1-9). The nano assembly device has sensitive < 1 > O2 self-reporting capability, improves tumor hypoxic microenvironment capability and exhausts GSH in tumor cells, further has a good anti-tumor effect and relatively high safety, and provides a new strategy for synergistic combination of a singlet oxygen detection probe 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 on tumor cells. In order to overcome this limitation, various strategies have been widely studied to deal with tumor hypoxia. Among them, the design of the oxygen delivery system aims to accurately deliver external oxygen to the interior of the tumor 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 state of the tumor microenvironment can be indirectly improved. However, these strategies face challenges in practical applications, such as the possibility of oxygen leakage of oxygen-carrying materials during transportation and the rapid consumption of supplemented oxygen at the tumor site.

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

[0005] In order to solve the existing problems of hypoxia in the tumor site, poor treatment effect in a high GSH environment and singlet oxygen in the PDT treatment process 1 The present invention provides a tumor microenvironment responsive nanoassembly and its preparation method and application. 1 The nanoassembly (DMCP nanoparticles) formed by DPA-MOF nanoparticles with real-time O2 monitoring function, manganese dioxide (MnO2) with GSH consumption and oxygen generation capabilities, and porphyrin photosensitizers can self-report the progress of PDT treatment. 1 O2 and regulate the tumor microenvironment to promote the therapeutic effect of PDT. MnO2 can catalyze the overexpressed H2O2 in the tumor site to produce oxygen and consume GSH in the tumor site. Under light conditions, the photosensitizer converts oxygen into a large amount of 1 O2, effectively kills tumor cells. At the same time, the DPA-MOF nanoparticles in this nanosystem 1 Real-time monitoring of O2 enables precise PDT treatment, effectively inducing tumor death without causing superimposed toxicity to normal cells. This self-reporting and self-enhancing nanoassembly provides a new drug delivery mode for multi-modal, 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 nanoassembly, wherein the nanoassembly is co-assembled by DPA-MOF nanoparticles, manganese dioxide and porphyrin photosensitizer through intermolecular forces and modified by a PEG modifier, wherein:

[0008] The mass ratio of the DPA-MOF nanoparticles, manganese dioxide and porphyrin photosensitizer is 1:0.1-10:0.1-15, and the mass ratio of the sum of the DPA-MOF nanoparticles, manganese dioxide and porphyrin photosensitizer 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 ultrasonication and then stirring in an oil bath.

[0010] In the above technical solution, during the preparation 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.

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

[0012] In the above technical solution, the stirring reaction condition is stirring the reaction at 800-1200 rpm for 5-6 hours.

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

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

[0015] Furthermore, the porphyrin photosensitizer is one or more of pyropheophorbide a, chlorophyll a, pheophorbide a, tetracarboxyphenylporphyrin, and dihydrochlorin 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 to 20,000.

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

[0020] The present invention also provides a method for preparing a tumor microenvironment responsive nanoassembly, comprising the following steps:

[0021] A certain amount of DPA-MOF nanoparticles is dispersed in deionized water to obtain a DPA-MOF nanoparticle solution, and then a poly(propyleneamine hydrochloride) solution is added, and the mixture is stirred at 800-1200 rpm for 1-2 hours to obtain a mixed solution; a potassium permanganate solution is added dropwise to the mixed solution, and the stirring is continued for 1-2 hours to obtain DM nanoparticles; the DM nanoparticles are then dispersed in a DMF solution, and a porphyrin photosensitizer solution is added dropwise to modify the DM nanoparticles, and the mixture is stirred at 800-1200 rpm for 10-14 hours to obtain DMC nanoparticles; the DMC nanoparticles are dispersed in water to obtain a DMC nanoparticle solution, and then an organic solvent containing a PEG modifier is added dropwise to the DMC nanoparticle solution under stirring at 800-1200 rpm, and the mixture is reacted for 12 hours. After the reaction, the organic solvent is removed to obtain DMCP nanoparticles, which are tumor microenvironment responsive nanoassemblies.

[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 the photosensitizer in an organic solvent with a concentration of 800 to 1000 nM.

[0024] In the above technical solution, the poly(propyleneamine hydrochloride) solution is prepared by dissolving poly(propyleneamine hydrochloride) in deionized water with a concentration of 12 to 15 mg / mL.

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

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

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

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

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

[0030] The process steps involved in the preparation method of the tumor microenvironment responsive nanoassembly of the present invention are all carried out at room temperature.

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

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

[0033] The present invention also provides an application of a tumor microenvironment responsive nanoassembly in the preparation of an injection, oral administration or local 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) used in the present invention is a new type of material with great potential, which has unique physical and chemical properties, including ultra-large specific surface area, high porosity, highly controllable size and excellent biocompatibility. These properties give Nano-MOF a strong gas adsorption ability, enabling it to efficiently capture gas during PDT treatment. 1 O2, significantly improves 1 The detection sensitivity of O2 also enables real-time monitoring during treatment. 1 O2 provides a possibility. More importantly, the Nano-MOF in the present invention is easy to modify. 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 integrate photosensitizers, tumor hypoxia alleviating agents and GSH depleting agents to achieve multi-component joint delivery, which can not only achieve 1 Real-time monitoring and precise control of O2 can 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 nanoassembly (DMCP nanoparticles) prepared by the present invention can be used for singlet oxygen detection and tumor microenvironment regulation to enhance the photodynamic therapy effect. 1 The timely capture and detection of O2 reflects the progress of PDT treatment and provides precise PDT treatment. MnO2 can increase the production of oxygen in the tumor site and deplete GSH, which can make the photosensitizer produce more under light conditions. 1 O2, while reducing 1O2 depletion enhances PDT-induced tumor death.

[0037] 3. A tumor microenvironment responsive nanoassembly prepared by the present invention achieves technical effects such as high sensitivity, good stability, and low toxic side effects, meeting the urgent clinical demand for high-efficiency and low-toxic preparations, and providing a new strategy for the synergistic assembly of singlet oxygen detection probes and other drugs, promoting the conceptual progress of using precision PDT to enhance anti-cancer mode. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 The scanning electron microscope images of DPA-MOF nanoparticles (a) and DMCP nanoparticles (b) obtained in Example 1.

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

[0040] Figure 3 This is the energy dispersive X-ray spectrum of the DMCP nanoparticles obtained in Example 1.

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

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

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

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

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

[0046] Fig. 9 This is a graph showing the GSH consumption of the DMCP nanoparticles obtained in Example 1.

[0047] Fig.10 The DMCP nanoparticles obtained in Example 1 under illumination (a) and non-illumination (b) conditions 1 O2 production situation.

[0048] Fig.11 The DMCP nanoparticles obtained in Example 1 were added with MnO2 under illumination (a) and non-illumination (b) conditions. 1 O2 production situation.

[0049] Fig.12 The fluorescence change of DPA-MOF nanoparticles obtained in Example 1 under the conditions of GSH (a) or GSH-free (b) is compared with that of DMCP nanoparticles under non-illumination conditions. 1 O2 detection research diagram (c).

[0050] Fig.13 The DMCP nanoparticles obtained in Example 1 were subjected to light exposure. 1 Detection sensitivity of O2 detection fluorescence change graph (a) and detection linear range graph (b).

[0051] Fig.14 The DMCP nanoparticles obtained in Example 1 are 1 Figure 2. Selectivity study of O2.

[0052] Fig.15 The safety test diagram of DPA-MOF nanoparticles (a) and DMCP nanoparticles (b) obtained in Example 1 on 4T1 cells.

[0053] Fig.16 The safety test diagram of DPA-MOF nanoparticles (a) and DMCP nanoparticles (b) obtained in Example 1 on MCF-7 cells.

[0054] Fig.17 The safety test diagram of DPA-MOF nanoparticles (a) and DMCP nanoparticles (b) obtained in Example 1 on 293T cells.

[0055] Fig.18 The uptake status and quantitative graph of DMCP nanoparticles obtained in Example 1 in 4T1 cells.

[0056] Fig.19 The uptake status and quantitative graph of DMCP nanoparticles obtained in Example 1 in MCF-7 cells.

[0057] Fig. 20 This is an investigation on the cytotoxicity of the DMCP nanoparticles obtained in Example 1 to 4T1 cells.

[0058] Fig.21 This is an investigation on the cytotoxicity of the DMCP nanoparticles obtained in Example 1 to MCF-7 cells.

[0059] Fig. 22 This is an investigation of the consumption of GSH in 4T1 cells by the DMCP nanoparticles obtained in Example 1.

[0060] Fig.23 This is an investigation of the consumption of GSH in MCF-7 cells by the DMCP nanoparticles obtained in Example 1.

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

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

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

[0064] Fig. 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, Shanghai Adamas Reagent Co., Ltd.), 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 and dispersed evenly by ultrasound. After being placed in an oil bath at 100 °C and stirred for 5 h, 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 to obtain a DPA-MOF nanoparticle solution, and then a poly(propyleneamine hydrochloride) (PAH) solution (1.25 mL, 14.4 mg / mL, the solvent was deionized water) was added, and the mixture was stirred and mixed at room temperature using a magnetic stirrer (stirring rate was 800 rpm) for 2 h. After that, a potassium permanganate (KMnO4) solution (1.25 mL, 8 mg / mL, the solvent was 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 a DMF solution (2 mL), and then a Ce6 solution (100 μL, 8 mM, the solvent was a DMF solution) was added dropwise to modify it. After stirring the reaction at 1000 rpm for 10 h, it was washed with water several 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): Disperse the above non-PEGylated DMC nanoparticles (4 mg) in water (2 mL). Add a DMF solution containing DSPE-PEG (1 mL, 2 mg / mL, molecular weight 2000) dropwise to the above solution under stirring (stirring rate 1000 rpm) and react for 12 hours. Finally, wash with water several times, remove the DMF solution by centrifugation, and obtain DMCP nanoparticles. The prepared DMCP nanoparticles are different from any other Zr-based MOF and have a unique 1 O2 detection and self-reporting capability.

[0070] Scanning electron microscopy of DPA-MOF nanoparticles and DMCP nanoparticles Figure 1 It can be seen that the prepared DPA-MOF nanoparticles are uniform in size, spherical, and well dispersed, with a particle size of about 120nm; after layer-by-layer modification, the surface of the DMCP nanoparticles becomes slightly rough, and the morphology of the nanoparticles does not change significantly, still a spherical structure with good dispersion, and a particle size of about 130nm. Transmission electron microscopy of DPA-MOF nanoparticles and DMCP nanoparticles Figure 2It can be seen that: DPA-MOF nanoparticles have a spherical structure of uniform size, a smooth surface, good dispersion, and a size of about 160nm; after layer-by-layer modification, DMCP nanoparticles have a wrinkled structure on the surface, which preliminarily proves that DMCP nanoparticles were successfully synthesized.

[0071] The average hydrated particle size of DPA-MOF and DMCP nanoparticles was measured by dynamic light scattering (DLS) technique, and the difference in surface charge of each nanoparticle (NPs) was 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 hydrated particle size of DPA-MOF nanoparticles is about 234 nm. After modification, the hydrated particle size of DMCP nanoparticles increases slightly 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. 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 skeleton, 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 elemental chemical state of DMCP nanoparticles. 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; the local high-resolution spectrum ( Figure 4 -b) The binding energies of 642.2 eV and 652.6 eV correspond to Mn(IV)2p 3 / 2 and Mn(IV)2p 1 / 2 This further demonstrates that MnO2 is successfully coated on the DPA-MOF surface.

[0077] The crystal characteristics of DPA-MOF and DMCP nanoparticles were analyzed by X-ray powder diffraction (XRD). Figure 5It can be seen that the position and intensity of the XRD diffraction peak of DPA-MOF nanoparticles are basically consistent with the simulated diffraction peak, which indicates that the DPA-MOF nanoparticles have been successfully prepared and have good crystallinity; in addition, the XRD diffraction peak of DMCP nanoparticles is basically consistent with the diffraction peak of DPA-MOF nanoparticles, indicating that the layer-by-layer modified MnO2, Ce6 and PEG have no obvious effect on the crystal form of 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 them to play their intended role. Therefore, the stability of the DMCP nanoparticles obtained above in different physiological solutions was evaluated. The DMCP nanoparticles were immersed in H2O, PBS buffer and DMEM complete medium for different time periods. The hydrated particle size of the DMCP nanoparticles was measured at 0, 12, 24, 48 and 72 hours. The results are shown in Table 1. Figure 6 It can be seen that after DMCP nanoparticles were treated with different physiological solutions for 72 hours, their hydrated particle size did not change significantly, indicating that 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 DMCP nanoparticles was investigated. The results are shown in Figure 7 It can be seen that the characteristic absorption peaks of photosensitizer Ce6 appear at 404nm, 503nm, 533nm, 607nm and 663nm, which are all caused by the π electron transition in the Ce6 structure; DPA-MOF has characteristic absorption peaks at 343nm, 360nm, 380nm and 400nm; in the UV-visible absorption spectrum of DMCP nanoparticles, there are characteristic absorption peaks of DPA-MOF and Ce6, which further confirms the successful preparation of nanoparticles; compared with free Ce6, the characteristic absorption peak of Ce6 in DMCP nanoparticles is red-shifted from 664nm to 674nm, which may be caused by the π-π stacking interaction of Ce6 molecules; in addition, the UV-visible absorption spectrum of DPA-MOF did not change significantly after loading MnO2. Similarly, the addition of PEG had no obvious effect on the UV-visible absorption spectrum of the nanoparticles.

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

[0083] Consumption capacity of DMCP nanoparticles on GSH in solution:

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

[0085] DMCP nanoparticles are produced in solution 1 O2's capabilities:

[0086] 1,3-Diphenylisobenzofuran (DPBF) is a commonly used commercial 1 O2 detection probe, has a characteristic absorption peak at 410nm. 1 After O2 undergoes irreversible oxidation reaction to generate the oxidation product DBB, the UV-visible absorption peak intensity at 410nm decreases. DPBF ethanol solution (5mM) was mixed with DMCP nanoparticle dispersion, and the mixture was aspirated at regular intervals under NIR laser irradiation. After filtering with a 0.22μm filter membrane, the change in absorbance of DPBF at the maximum absorption wavelength was measured. At the same time, the UV-visible spectrum tested at the same time under non-light conditions was used as a control. The results are shown in Fig.10 , it can be seen that: under lighting conditions, DPBF is 410nm The absorbance value at 100 nm showed a significant downward trend, indicating that Ce6 loaded in DMCP nanoparticles continuously generated1 O2; in contrast, under non-illumination conditions, the absorption peak of DPBF hardly changes. This proves that under the excitation of light of appropriate wavelength, DMCP nanoparticles have good 1 O2 production capacity.

[0087] After adding MnO2, the 1 The ability to produce O2 can be enhanced. The DMCP nanoparticles aqueous dispersion was added to the TME simulated solution (composed of PBS buffer solution with a pH of 6.5 and 10 mM GSH solution), and the changes in the UV-visible absorption spectrum of the DMCP nanoparticles after different illumination times were observed to determine whether the DMCP nanoparticles have the ability to produce and detect O2. 1 At the same time, the UV-visible absorption spectrum of the system under non-light conditions at the same time intervals was used as a control, and the results are shown in Fig.11 It can be seen that with the increase of illumination time, the characteristic absorption peak intensity of the anthracene ring in the DMCP nanoparticles is constantly decreasing, indicating that under the excitation of NIR laser, Ce6 in the nanoparticles produces 1 O2 was captured by the probe DPA-MOF, causing its absorbance to decrease. However, under non-illumination conditions, the intensity of the characteristic absorption peak of the anthracene ring did not change significantly, indicating that no oxygen was generated in the system under light-shielding conditions. 1 O2, and also shows that DMCP nanoparticles have good stability. The above results can preliminarily judge that DMCP nanoparticles have good 1 O2 generation and detection capabilities.

[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 (i.e., the system only contained free Ce6 as 1 O2 source, DPA-MOF 1 O2 detection probe) is produced in a simulated tumor microenvironment 1 In the case of O2, the content of Ce6 in the two systems was controlled to be the same, and the results are shown in Fig.12 -a,b, it can be seen that: in the system containing DMCP nanoparticles, with the increase of illumination time, the characteristic absorption peak of the anthracene ring gradually decreases, indicating that the system is constantly generating 1 O2; In the system without MnO2, the UV absorption peak of the anthracene ring decreased to a certain extent within 15 minutes of illumination, but there was no obvious change in the absorbance of DPA during the subsequent illumination time. The change in the intensity of the anthracene ring absorption peak in the two systems also represents the difference between the two systems. 1 The O2 content was significantly different, and DMCP nanoparticles produced more O2 than free Ce6. 1O2 is more, this is because MnO2 in DMCP nanoparticles plays an important role in simulating TME, MnO2 consumes GSH and reduces the effect of GSH on 1 The consumption of O2 indirectly increases the 1 O2 content.

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

[0090] DPA-MOF can emit blue fluorescence when excited by appropriate wavelengths. 1 After O2 is removed, the corresponding endoperoxide will be generated, resulting in quenching of blue fluorescence. Using this fluorescence response mechanism, the change of DPA-MOF's own fluorescence can be used to achieve 1 Therefore, fluorescence spectroscopy was used to obtain the fluorescence emission spectra of the system containing DMCP nanoparticles and the system without MnO2, and the fluorescence response ability of the nanofluorescent probe was combined to investigate the enhancement of DMCP nanoparticles. 1 O2 generation and detection 1 O2's capabilities.

[0091] The DMCP nanoparticles aqueous dispersion was added into the TME simulated solution (25 μg mL -1 ), after incubation for 20 min, 655 nm NIR laser was used for irradiation, 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 Fig.12 -c, it can be seen that: in the DMCP nanoparticle system containing MnO2, with the extension of illumination time, the fluorescence intensity of DPA-MOF gradually decreases, indicating that DPA-MOF successfully captures the generated in the system 1 O2 forms the corresponding endoperoxide, which leads to the quenching of its own fluorescence. In the system without MnO2, the fluorescence intensity of DPA-MOF only decreases to a small extent when the laser is irradiated for the same time, indicating that the fluorescence intensity of DPA-MOF in the system 1 The O2 content is low. This is because there is a high concentration of GSH in the system, and the reduced GSH will consume the 1 O2; Under non-illumination conditions, the fluorescence emission spectrum of DPA-MOF has almost no change, indicating that there is no 1 O2 is generated. This also proves that the change in DPA-MOF fluorescence intensity has nothing to do with laser irradiation. 1 O2, the emission peak will drop significantly.

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

[0093] Study on DMCP nanoparticles as nanofluorescent probes 1 O2 detection sensitivity, using NaClO+H2O2 system to generate exogenous 1 O2, DMCP nanoparticles, NaClO (10mM) 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 5mM) were added to the reaction system in sequence. After the reaction system was reacted for 15 minutes at room temperature without light, the changes in the fluorescence emission spectrum of DPA-MOF were measured. The fluorescence emission intensity ratio (F0 / F) of DPA-MOF was used for quantitative analysis 1 The linearity and range were determined, and the detection limit (LOD) was calculated, where F0 represents the initial emission intensity of DPA without H2O2, and F represents the fluorescence emission intensity of DPA after adding different concentrations of H2O2. Fig.13 , it can be seen that: with 1 As the O2 concentration increases, the fluorescence emission intensity of DPA-MOF decreases. 1 When the O2 concentration increases from 0 to 5mM, the F0 / F value increases from 1 to 2.63. In the range of 0.2-3mM, 1 O2 concentration and F0 / F value showed a good linear relationship (r = 0.996); DMCP nanoparticles 1 The detection limit of O2 was approximately 79 μM.

[0094] DMCP nanoparticles in solution 1 Study on detection selectivity of O2:

[0095] ROS are natural byproducts of the body's normal oxygen metabolism, mainly including superoxide anion free radicals (·O2 - ), hydrogen peroxide (H2O2), hydroxyl radical (·OH) and 1 In order to investigate the effect of DMCP nanoparticles on free radicals and non-free radical substances. 1 To investigate the specificity of O2 detection, we determined the effect of DMCP nanoparticles on the oxygen concentration in the presence of other interfering substances such as ROS or ions. 1 The selectivity of O2 was determined by setting a blank group (DMCP nanoparticle dispersion without ROS) as a control to control the concentration of ROS. After incubating DMCP nanoparticles with different types of ROS for 15 minutes, the changes in the fluorescence emission spectrum of DMCP nanoparticles were measured. The concentration of ROS and interfering ions used was 5mM, and the preparation method was as follows:

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

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

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

[0099] ·OH: 5mM FeSO4·7H2O+5mM H2O2;

[0100] C1O - : 100 mM NaClO diluted to 5 mM.

[0101] The fluorescence intensity ratio (F / F0) after the reaction of DMCP nanoparticles with various ROS substances was measured. The results are shown in Fig.14 , it can be seen that: 1 In the presence of O2, the F / F0 of DMCP nanoparticles decreased significantly; however, in the presence of other interfering substances, the F / F0 of DMCP nanoparticles did not change significantly, which proved that DMCP nanoparticles 1 O2 detection has good selectivity.

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

[0103] 4T1 cells, MCF-7 cells and 293T cells in good growth state were diluted with culture medium to prepare cell suspension at 3×10 3 The cells were inoculated at a density of 100 μL / well in a 96-well plate, and 200 μL of cell suspension was added to each well. Four replicate wells were set for each group of samples and cultured in a cell culture incubator for 12 h to allow the cells to adhere to the wall and grow. The original culture medium in the 96-well plate was replaced with suspensions containing different concentrations of Ce6, DMCP nanoparticles or DPA-MOF. In the Ce6 and DMCP nanoparticle groups, the Ce6 content of the two was controlled to be the same, and the Ce6 content was set to 0, 1, 2, 4, 8, 16 and 20 μg mL -1 , the concentration of DPA-MOF was set to 0, 5, 10, 20, 40, 80, and 100 μg mL -1 In order to eliminate the interference of background, a blank control group containing only PBS buffer solution was set up. After 12 h of culture, the culture medium containing Ce6 solution, DPA-MOF and DMCP nanoparticles was removed, and after washing with PBS buffer solution, 100 μL of culture medium was added to each well, and 20 μL of MTT solution (5 mg mL -1 ), and then after 4 hours of continuous culture, the culture medium was discarded, 150 μL DMSO was added to each well, and the cells were gently shaken for 5 minutes using a plate shaker to fully dissolve the generated formazan. Finally, the absorbance at 490 nm was measured using an ELISA reader, and the cell survival rate was calculated. Figure 15-17 It can be seen that: without NIR laser irradiation, with the increase of Ce6 solution, DPA-MOF and DMCP nanoparticle concentrations, the survival rate of the three cells can be maintained above 70%. This shows that free Ce6 and the prepared nanofluorescent probes DPA-MOF and DMCP nanoparticles have low toxicity under non-light conditions, have little effect 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] The 4T1 cells with good growth status were digested to prepare cell suspension and inoculated into a 24-well plate with slides at a cell density of 5×10 4 / well, culture for 12h, so that the cells grow on the crawling slice. After that, the original culture medium was replaced with a culture medium containing DMCP nanoparticles, and incubation was continued for 0, 4, 8, 12 and 16h. Then, the culture medium was removed and washed three times with PBS buffer solution to remove excess DMCP nanoparticles. Then, the cells were fixed in the dark for 30min with 4% tissue cell fixative, and then washed three times with PBS buffer solution to remove excess fixative. Finally, the cell nucleus staining agent Hoechst33324 was added, and after staining in the dark for 10min, the crawling slice was taken out and fixed on a glass slide with a small amount of anti-fluorescence attenuation mounting agent. The uptake of DMCP nanoparticles by 4T1 cells was observed by observing blue fluorescence and red fluorescence by CLSM. Among them, the blue fluorescence emitted by Hoechst33324 can be used to determine the position of the cell nucleus, and the Ce6 loaded in the DMCP nanoparticles can emit red fluorescence under the excitation of laser of appropriate wavelength. According to the change of the intensity of the 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 medium containing DMCP nanoparticles was added after incubation of MCF-7 cells for 24 h, the rest of the steps were the same as the uptake experimental steps of 4T1 cells. The average fluorescence intensity of the obtained fluorescence images was analyzed using Image J software. The results are shown in Fig.18 and Fig.19 .

[0106] CLSM images after incubation of DMCP nanoparticles with 4T1 cells for 0, 4, 8, 12 and 16 h ( Fig.18 -a) shows that the cell nucleus is stained blue, and the red fluorescence comes from the DMCP nanoparticles, which surround the cell nucleus. This shows that the DMCP nanoparticles are successfully taken up by 4T1 cells. Fig.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 hours, the red fluorescence intensity in the cells gradually increased with the extension of incubation time and reached a balance state at 12 hours. It can be concluded that the optimal uptake time of DMCP nanoparticles by 4T1 cells is 12 hours.

[0107] CLSM images of DMCP nanoparticles after incubation with MCF-7 cells for 0, 4, 8, 12 and 16 h ( Fig.19 -a) can confirm that DMCP nanoparticles have been effectively taken up by MCF-7 cells. Fig.19 -b) By comparison, it can be seen that after MCF-7 cells were co-incubated with DMCP nanoparticles for 12 hours, the red fluorescence in the cells was the strongest, indicating that the optimal uptake time for MCF-7 cells to DMCP nanoparticles was 12 hours.

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

[0109] The MTT experiment was used to determine the PDT anti-tumor effect of the DMCP nanoparticles under 655nm NIR laser irradiation. 4T1 cells and MCF-7 cells in good cell state were digested to prepare cell suspensions. 3 The cells were inoculated in a 96-well plate at a density of 100 μg / well. After 12 h of culture, the original culture medium was discarded and different concentrations of Ce6 and DMCP nanoparticle suspensions were added. After laser irradiation for different times (0, 5, 10 and 20 min), the cells were incubated for 24 h, and then the PDT treatment effects of each experimental group were compared by MTT method. At the same time, the cytotoxicity of DMCP nanoparticles on 4T1 cells and MCF-7 was evaluated. The results are shown in Fig. 20 and Fig.21 . Fig. 20 To show the changes in the survival rate of 4T1 cells after laser irradiation after treatment with different concentrations of Ce6 and DMCP nanoparticles. Fig.21 Figure 3 shows the changes in the survival rate of MCF-7 cells after the same treatment. It can be seen that: under the same concentration of Ce6 or DMCP nanoparticles, with the increase of illumination time, the cell viability gradually decreased; after 20 minutes of illumination, the inhibition rate of tumor cells in the group containing DMCP nanoparticles was the highest, and there was no obvious damage to the cells in the group without DMCP nanoparticles, indicating that the PDT killing effect of cells treated with DMCP nanoparticles was more obvious. This is because the MnO2 loaded in the DMCP nanoparticles can consume the overexpressed GSH in the TME and reduce the effect of GSH on the tumor cells. 1 O2 consumption makes the cells 1 The O2 content increases, thereby amplifying the therapeutic effect of PDT.

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

[0111] The GSH detection kit was used to further study the consumption of intracellular GSH by DMCP nanoparticles. 4T1 cells and MCF-7 cells in good condition were digested into cell suspension and inoculated in 6-well plates at a density of 2×10 5 / well. After incubation for 12 hours, the original culture medium was replaced with culture medium containing different concentrations of Ce6 or DMCP nanoparticles (the Ce6 content of the two was controlled to be the same). After incubation for another 12 hours, the culture medium was removed, washed three 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 1mL of reagent 1 (GSH extract), repeatedly frozen and thawed 2 to 3 times (freeze in liquid nitrogen and dissolve in a 37°C water bath), and the supernatant was collected after centrifugation at 10,000rpm for 10 minutes. According to the ratio provided in the kit instructions, 20μL of sample, 140μL of reagent 2 (GSH analysis buffer) and 40μL of reagent 3 (color developer DTNB) were added to the 96-well plate in sequence. After standing for 2 minutes, the absorbance value at 405nm was measured using an enzyme reader. The determination process of the GSH content in MCF-7 cells is consistent with the operation process of 4T1 cells, except that the culture medium containing DMCP nanoparticles was added after 24 hours of culture. The above experimental results are shown in Fig. 22 and Fig.23 It can be seen that different concentrations of Ce6 have no significant effect on the level of GSH in 4T1 cells. After the cells are incubated with DMCP nanoparticles, the content of GSH in the cells gradually decreases with the increase of the concentration of Ce6 solution and DMCP nanoparticles. When the concentration of Ce6 solution and DMCP nanoparticles is 1.09 μg mL -1 (where Ce6 concentration is 200 ng·mL -1 ), the content of GSH in 4T1 cells can be reduced to about 48%; after being treated with different concentrations of Ce6 or DMCP nanoparticles, the changes in the level of GSH in MCF-7 cells are similar to those in 4T1 cells. After incubation of Ce6 with cells, the level of GSH in cells did not change significantly, while the level of GSH in cells treated with DMCP nanoparticles was significantly reduced. The above experimental results show that DMCP nanoparticles can effectively remove overexpressed GSH in tumor cells and reduce the content of GSH.

[0112] DMCP nanoparticles produced in vitro 1 O2 Research:

[0113] 4T1 or MCF-7 cells in good condition were cultured at 5 × 10 4The cells were seeded at a density of 100 μg / well in a 24-well plate. After the cells adhered to the wall, Ce6 or DMCP nanoparticle suspension was added (the Ce6 content in the two suspensions was controlled to be the same). After incubation for 12 hours, the culture medium was aspirated and washed three times with PBS buffer solution to remove the Ce6 solution and DMCP nanoparticles that were not taken up. Then, DCFH-DA solution (20 μM) was added, incubated at 37°C for 30 minutes, and then irradiated with a 655nm laser for 20 minutes. Finally, an inverted fluorescence microscope was used to perform fluorescence imaging of the cells after different illumination times. The results are shown in Figure 2. Fig.24 and Fig.25 It can be seen that compared with the control group, the Ce6 and DMCP nanoparticle groups in 4T1 cells and MCF-7 cells showed green fluorescence after laser irradiation, indicating that Ce6 solution or DMCP nanoparticles produced green fluorescence in the cells under NIR laser excitation. 1 O 2； 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, this is because the MnO2 contained in the DMCP nanoparticles consumes GSH and reduces the amount of GSH on 1 O2 consumption. This further proves that DMCP nanoparticles have the potential to enhance the efficacy of PDT.

[0114] DMCP nanoparticles in vitro 1 O2 Detection Research:

[0115] In vitro DMCP nanoparticle detection 1 O2 capacity. 4T1 or MCF-7 cells in good condition were cultured at 5×10 4 The cells were seeded at a density of 100 μg / well in a 24-well plate. After the cells adhered to the wall, 1 mL of DMCP nanoparticles (2 μg mL -1 ) suspension. After incubation for 12 h, the cells were irradiated with 655 nm laser for different time periods (0, 5, 10 and 20 min), then the culture medium was aspirated and washed three times with PBS buffer solution. Finally, fluorescence imaging of the cells after different illumination times was performed using an inverted fluorescence microscope. The fluorescence intensity changes of DPA itself reflected the changes during PDT treatment. 1 The generation of O2 is shown in Fig.26 and Fig. 27 The changes in the fluorescence intensity of DPA in cells under different illumination times show that in the two tumor cells treated with DMCP nanoparticles, the blue fluorescence of DPA gradually weakened with the increase of illumination time, indicating that DMCP nanoparticles can detect the blue fluorescence of DPA produced during PDT by changing the fluorescence of the probe itself. 1 O2 is tested.

Claims

1. A tumor microenvironment responsive nanoassembly, characterized in that: The nanoassembly is co-assembled by DPA-MOF nanoparticles, manganese dioxide and porphyrin photosensitizer through intermolecular forces, and is modified by a PEG modifier; wherein, The mass ratio of the DPA-MOF nanoparticles, manganese dioxide and porphyrin photosensitizer is 1:0.1-10:0.1-15, and the mass ratio of the sum of the DPA-MOF nanoparticles, manganese dioxide and porphyrin photosensitizer to the PEG modifier is 1:0.1-9.

2. The nanoassembly according to claim 1, characterized in that: The porphyrin photosensitizer is one or more of pyropheophorbide a, chlorophyll a, pheophorbide a, tetracarboxyphenylporphyrin, and dihydrochlorin e6.

3. The nanoassembly according to claim 1, characterized in that: The intermolecular force is an electrostatic force.

4. The nanoassembly according to claim 1, characterized in that: 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.

5. A method for preparing a tumor microenvironment responsive nanoassembly according to any one of claims 1 to 4, characterized in that: The steps include: A certain amount of DPA-MOF nanoparticles is dispersed in deionized water to obtain a DPA-MOF nanoparticle solution, and then a poly(propyleneamine hydrochloride) solution is added, and the mixture is stirred at 800-1200 rpm for 1-2 hours to obtain a mixed solution; a potassium permanganate solution is added dropwise to the mixed solution, and the mixture is stirred for 1-2 hours to obtain DM nanoparticles; the DM nanoparticles are dispersed in a DMF solution, and a porphyrin photosensitizer solution is added dropwise to modify the DM nanoparticles, and the mixture is stirred at 800-1200 rpm for 10-14 hours to obtain DMC nanoparticles; the DMC nanoparticles are dispersed in water to obtain a DMC nanoparticle solution, and then an organic solvent containing a PEG modifier is added dropwise to the DMC nanoparticle solution under stirring at 800-1200 rpm, and the mixture is reacted for 12 hours. After the reaction, the organic solvent is removed to obtain DMCP nanoparticles, which are tumor microenvironment responsive nanoassemblies.

6. The method according to claim 5, characterized in that: The organic solvent is one or more of N,N-dimethylformamide, dimethyl sulfoxide and tetrahydrofuran.

7. Use of the tumor microenvironment responsive nanoassembly according to any one of claims 1 to 4 or the nanoassembly obtained by the preparation method according to claim 5 or 6 in the preparation of a drug delivery system.

8. Use of the tumor microenvironment responsive nanoassembly according to any one of claims 1 to 4 or the nanoassembly obtained by the preparation method according to claim 5 or 6 in the preparation of anti-tumor drugs.

9. Use of the tumor microenvironment responsive nanoassembly according to any one of claims 1 to 4 or the nanoassembly obtained by the preparation method according to claim 5 or 6 in the preparation of an injection, oral administration or local administration system.

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