A nanoparticle with tumor microenvironment regulation, multifunctional nanoparticle and preparation method and application thereof

Abstract

The application discloses a kind of nanoparticles with tumor microenvironment regulation, multifunctional nanoparticles and preparation method and application thereof, including core, the core is Prussian blue analogue nanoparticle;Protective layer, covers the surface of the core, the protective layer has the characteristics of degradation in acidic environment;And, targeting agent connected on protective layer.The application has the ability to respond and adjust to tumor microenvironment (TME), can realize photothermal therapy and photodynamic therapy under the guidance of two modes of imaging, such as photothermal and nuclear magnetic, under single NIR laser irradiation.The material has the advantages of simple preparation process, low cost, good biocompatibility, tumor microenvironment response and modification, can realize targeted, non-invasive, efficient killing of tumor cells, so as to have broad clinical application prospect in tumor treatment.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of organic compound synthesis, and particularly relates to a nanoparticle with tumor microenvironment regulation, a multifunctional nanoparticle and a preparation method and application thereof. BACKGROUND

[0002] Cancer is one of the global health problems, and has a high incidence and mortality rate. A nano-therapy platform has become one of the strategies for cancer treatment, which combines multiple anti-tumor therapies with multi-modal imaging to monitor tumor lesions, reduce drug resistance toxicity, and improve treatment effect. Therefore, nano-medicine provides a new research direction for the treatment of cancer. Photothermal therapy (PTT) and photodynamic therapy (PDT) are concerned due to their small invasiveness and strong spatiotemporal controllability (Nano-Micro Lett. 2018, 10, 74; Chem. Eng. J. 2020, 388, 124212; Colloid Surf. B-Biointerfaces 2020, 194, 111213). PTT activates a photothermal agent (PTA) by near-infrared light (NIR) to ablate tumors by raising the temperature of tumor tissues (Photobiol. B-Biol. 2022, 228, 112407; Small 2020, 16, 2003707). PDT kills tumor cells by generating cytotoxic reactive oxygen species through photosensitizers (PS) under the presence of oxygen (Eur. J. Med. Chem. 2021, 225, 113770; ACS Nano 2018, 12, 11446).

[0003] Hypoxia is a prominent feature of the tumor microenvironment (TME) as it limits the production of reactive oxygen species (ROS) by tumor cells, which can reduce the therapeutic efficacy of photodynamic therapy (PDT) (Cancer Lett. 2020, 492, 116; Adv. Sci. 2022, 9, 2203292). To overcome this challenge, research efforts have proposed various strategies to increase the oxygen concentration within the tumor. One strategy is to deliver oxygen carriers, such as perfluorocarbons and hemoglobin (Adv. Funct. Mater. 2017, 27, 1703197; Adv. Funct. Mater. 2019, 29, 1806199) to the tumor site; while another approach is to introduce peroxidase-mimicking nanoparticles that catalyze the in situ generation of oxygen from hydrogen peroxide at the tumor site. Nanoszymes, a type of nanomaterial that is more stable than natural enzymes, have been widely used in catalytic oxygen production. Some platinum (ACSAppl. Mater. Interfaces 2019, 11, 17215) and manganese (Sensors 2020, 20, 6905; J. Mater. Chem. B 2020, 8, 7121.) based nanoszymes exhibit peroxidase-like activity, which can facilitate oxygen production and improve the efficacy of PDT in hypoxic TME.

[0004] In addition to hypoxia, solid tumors also exhibit elevated levels of glutathione (GSH). GSH is an antioxidant that can reduce the cytotoxic ROS induced by PDT, leading to a decrease in PDT efficacy (Small 2022, 18, 2105465). Some polyvalent metal ions have been shown to have the ability to scavenge excess GSH in tumors (ACS Appl. Mater. Interfaces 2020, 12, 17254). For example, iridium oxide nanoparticles (IrOx) can exhibit excellent GSH consumption ability by transitioning between Ir 3+ and Ir 4+ To address the complex and challenging tumor microenvironment (TME) of solid tumors and improve the efficacy of photodynamic therapy (PDT), strategies to overcome hypoxia and GSH scavenging can be combined on a nanoplatform for optimization.

[0005] However, the prior art still has the following disadvantages: (1) some nanomaterials have single treatment effect and poor effect; (2) some nanomaterials lack active targeting effect and cannot specifically recognize cancer cells; (3) some materials only have treatment effect and lack imaging diagnosis capability, making it difficult to accurately combine imaging to guide treatment; (4) most nanomaterials cannot respond to tumor microenvironment and cannot achieve controllable treatment; (5) most nanomaterials lack the ability to modify the tumor microenvironment, making it difficult to effectively achieve photodynamic therapy. SUMMARY

[0006] This section is intended to summarize some aspects of the embodiments of the present application and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of the specification to avoid obscuring the purpose of this section, the abstract and the title, and such simplifications or omissions cannot be used to limit the scope of the present application.

[0007] In view of the above and / or problems existing in the prior art, the present application is proposed.

[0008] One of the purposes of the present application is to provide a kind of nanometer particle with tumor microenvironment regulation, with the ability to respond and adjust to tumor microenvironment (TME), the material has the advantages of simple preparation process, low cost, good biocompatibility, pH response and modification, can realize targeted, non-invasive, efficient killing of tumor cells.

[0009] The so-called "tumor microenvironment" herein refers to the microenvironment around tumor cells, including blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, signal molecules and extracellular matrix, etc. The characteristics of tumor microenvironment include overall hypoxia, acidification, interstitial high pressure, vascular high permeability, inflammatory reactivity and immunosuppression, etc.

[0010] The so-called "pH response" herein refers to a kind of nanomaterial that can respond to changes in pH value in the environment. These materials usually have special structure or composition, so that they change physically or chemically under different pH conditions, thereby changing their properties or functions.

[0011] To solve the above technical problems, the present application provides the following technical solutions: a Prussian blue analogue nanoparticle, the Prussian blue analogue nanoparticle is a Prussian blue analogue doped with Mn and Ir ions, which is obtained by reacting K3[Fe(CN)6] with a solution containing Fe 2+ , Mn 2+ , Ir 3+ ;

[0012] K3[Fe(CN)6] and Fe 2+ , Mn 2+ , Ir3+ at a molar ratio of 10:3 to 5:2 to 4:2 to 4.

[0013] As referred to herein, a "Prussian blue analog" is a typical coordination framework material, which can be represented by the general formula A x M1[M2(CN)6] y ·zH2O, wherein M1 / M2 is a transition metal connected by cyano groups, and A is a cation embedded in the interstitial space of the PBA framework. For example, the Prussian blue analog employed in the present application has a structure represented by Ir x Mn y [Fe(CN)6].

[0014] As a preferred embodiment of the Prussian blue analog nanoparticle of the present application, the Prussian blue analog nanoparticle has a hydrodynamic diameter of 300 to 350 nm and a Zeta potential of -10 to -20 mV.

[0015] As referred to herein, the "hydrodynamic diameter" refers to the particle size of a nanoparticle calculated by means of Dynamic Light Scattering (DLS) technology, in which a laser is used to irradiate the particles, and the intensity fluctuation of scattered light is analyzed, thereby calculating the particle size of the nanoparticles. The overall particle size distribution of the nanomaterial is detected. It should be noted that the DLS does not measure the true size of the nanoparticles, but the hydrodynamic diameter of the nanoparticles in solution; compared with the data measured by an electron microscope, the DLS measurement result is usually slightly larger.

[0016] Another object of the present application is to provide a nanoparticle with tumor microenvironment regulation, comprising,

[0017] a core, wherein the core is a Prussian blue analog nanoparticle;

[0018] a protective layer covering the surface of the core, wherein the protective layer has a property of degrading in an acidic environment; and a targeting agent connected to the protective layer.

[0019] As a preferred embodiment of the nanoparticle with tumor microenvironment regulation of the present application, the protective layer comprises one or more of pH-sensitive calcium carbonate, calcium phosphate, phospholipid, polymer, and gel.

[0020] The protective layer covering the surface of the core increases the hydrodynamic diameter of the nanoparticle to 310 to 370 nm and decreases the Zeta potential to -20 to -40 mV.

[0021] As a preferred solution of the tumor microenvironment-regulated nanoparticle of the present application, wherein: the targeting agent is a tumor cell-targeting aptamer, the targeting agent connected to the protective layer increases the hydrodynamic diameter of the nanoparticle to 330-450 nm, and the Zeta potential is -10 to -20 mV.

[0022] As a preferred solution of the tumor microenvironment-regulated nanoparticle of the present application, wherein: the aptamer comprises sequences targeting transferrin receptors and epithelial cell adhesion molecules expressed on tumor cells; the sequence of the aptamer targeting transferrin receptors on tumor cells is GCGCGGTACCGCGC, and the sequence of the aptamer targeting epithelial cell adhesion molecules on tumor cells is ACGGAGGTTGCGTCCGT.

[0023] As a preferred solution of the tumor microenvironment-regulated nanoparticle of the present application, wherein: the tumor cell-targeting aptamer is modified with biotin and / or labeled with ROX fluorescence.

[0024] Another object of the present application is to provide a preparation method of the tumor microenvironment-regulated nanoparticle as described above, comprising,

[0025] synthesizing a core of the nanoparticle;

[0026] synthesizing a protective layer to cover the core with the protective layer;

[0027] connecting a targeting agent to the protective layer.

[0028] As a preferred solution of the preparation method of the tumor microenvironment-regulated nanoparticle of the present application, wherein: the core of the synthesized nanoparticle, an aqueous solution of K3[Fe(CN)6] is added to a solution containing Fe 2+ , Mn 2+ , and Ir 3+ , mixed, reacted in the presence of citric acid, centrifuged, washed, and dried to obtain Prussian blue analog nanoparticles PMI;

[0029] wherein the molar ratio of K3[Fe(CN)6] to Fe 2+ , Mn 2+ , and Ir 3+ is 10:3-5:2-4:2-4.

[0030] As a preferred solution of the preparation method of the tumor microenvironment-regulated nanoparticle of the present application, wherein: the Fe 2+ , Mn 2+ , and Ir 3+ are each selected from one of their corresponding sulfate, hydrochloride, and phosphate.

[0031] As a preferred scheme of the preparation method of the tumor microenvironment regulating nanoparticle of the present application, wherein: the inner core of the synthetic nanoparticle, in particular:

[0032] Under acidic conditions, in the aqueous solution of K3[Fe(CN)6], iron salt, manganese salt, iridium salt is added, the temperature is controlled at 40-80℃, the stirring speed is 800-1400rpm, and the solution is stirred until it is blue; solid-liquid separation and drying obtain Prussian blue analog nanoparticles (PMI).

[0033] As a preferred scheme of the preparation method of the tumor microenvironment regulating nanoparticle of the present application, wherein: the temperature is 50-70℃, and the stirring speed is 1000-1300rpm.

[0034] As a preferred scheme of the preparation method of the tumor microenvironment regulating nanoparticle of the present application, wherein: the solid-liquid separation method is high-speed centrifugation, and the centrifugal speed is 12000-16000rpm, preferably 14000rpm.

[0035] As a preferred scheme of the preparation method of the tumor microenvironment regulating nanoparticle of the present application, wherein: the washing is performed 2-8 times with deionized water.

[0036] As a preferred scheme of the preparation method of the tumor microenvironment regulating nanoparticle of the present application, wherein: the drying is performed in a vacuum dryer, the drying temperature is 50-80℃, and the drying time is 12-24h; preferably, the drying temperature is 60℃, and the drying time is 24h.

[0037] As a preferred scheme of the preparation method of the tumor microenvironment regulating nanoparticle of the present application, wherein: the synthesis of the protective layer is to cover the inner core with the protective layer, sodium carbonate solution is added to the calcium chloride solution uniformly dispersed with PMI to obtain PMIC, wherein the mass ratio of PMI to calcium chloride is 1:1-2.

[0038] As a preferred scheme of the preparation method of the tumor microenvironment regulating nanoparticle of the present application, wherein: the synthesis of the protective layer is to cover the inner core with the protective layer, sodium carbonate solution is added to the calcium chloride solution uniformly dispersed with PMI to obtain PMIC, wherein the mass ratio of PMI to calcium chloride is 1:1-2.

[0039] Under the condition of vigorous stirring, the PMI-containing calcium chloride solution is added to the cyclohexane, Triton X-100 and 1-hexanol solution, and then the sodium carbonate solution is added, and stirred for 8-24h; solid-liquid separation to obtain PMI@CaCO3(PMIC) nanoparticles.

[0040] As a preferred scheme of the preparation method of the tumor microenvironment regulating nanoparticle of the present application, wherein: the molar ratio of the calcium chloride to the sodium carbonate is 1:8-12.

[0041] As a preferred solution of the preparation method of the tumor microenvironment regulating nanoparticle of the present application, wherein: cyclohexane: Triton X-100: 1-hexanol: PMI = 7.5 mL: 1.5-2 mL: 1.5-2 mL: 2-3 mg by mass volume.

[0042] As a preferred solution of the preparation method of the tumor microenvironment regulating nanoparticle of the present application, wherein: the solid-liquid separation adopts centrifugal separation, the centrifugal speed is 600-10000 rpm, preferably 8000-9000 rpm, and more preferably 8500 rpm.

[0043] As a preferred solution of the preparation method of the tumor microenvironment regulating nanoparticle of the present application, wherein: after the solid-liquid separation, the PMIC is washed with anhydrous ethanol and deionized water for 3-5 times, respectively.

[0044] As a preferred solution of the preparation method of the tumor microenvironment regulating nanoparticle of the present application, wherein: the target agent is connected to the protective layer, PMIC is added to the PBS solution containing acetamide (BSA) to obtain PMIC@BSA, then streptavidin is added to the mixed solution of PMIC@BSA and glutaraldehyde for incubation, the nucleic acid aptamer is annealed using PBS containing magnesium chloride, and the three-dimensional structure is folded, and the PMIC is prepared after the nucleic acid aptamer is mixed with PMIC@BSA in PBS. Apt ; wherein the mass ratio of the nucleic acid aptamer to PMIC is 80:1-2.

[0045] As a preferred solution of the preparation method of the tumor microenvironment regulating nanoparticle of the present application, wherein: by mass, BSA: PMIC = 1: 150-250.

[0046] As a preferred solution of the preparation method of the tumor microenvironment regulating nanoparticle of the present application, wherein: the streptavidin is added to the mixed solution of PMIC@BSA and glutaraldehyde for incubation, the streptavidin solution is added to the PMIC@BSA solution for incubation, the supernatant is removed by centrifugation again, and the PMIC@BSA loaded with avidin is fully mixed with glutaraldehyde in the PBS solution.

[0047] As a preferred solution of the preparation method of the tumor microenvironment regulating nanoparticle of the present application, wherein: the mass ratio of the streptavidin to PMIC@BSA is 150-250:1.

[0048] As a preferred solution of the preparation method of the tumor microenvironment regulating nanoparticle of the present application, wherein: the mass ratio of the PMIC@BSA loaded with avidin to glutaraldehyde is 1:1-3.

[0049] As a preferred scheme of the preparation method of the tumor microenvironment-regulated nanoparticle of the present application, wherein: the annealing is performed by using a PCR instrument, the annealing temperature is 95℃ for 5 min, and the cooling is performed slowly to 22℃ for 10 min.

[0050] The "annealing" referred to herein refers to a process of breaking all hydrogen bonds of the aptamer by heating, thereby destroying all secondary structures in each aptamer, then promoting the hybridization reaction by slow cooling, and finally forming new hydrogen bonds between complementary sequences.

[0051] Another object of the present application is to provide a multifunctional nanoparticle, comprising,

[0052] The tumor microenvironment-regulated nanoparticle as described above; and,

[0053] The photosensitizer loaded on the nanoparticle.

[0054] As a preferred scheme of the multifunctional nanoparticle of the present application, wherein: the absorption spectrum of the photosensitizer overlaps with the absorption spectrum of the nanoparticle.

[0055] As a preferred scheme of the multifunctional nanoparticle of the present application, wherein: the hydrodynamic diameter of the multifunctional nanoparticle is 380-460 nm, and the Zeta potential is -10 to -20 mV.

[0056] As a preferred scheme of the multifunctional nanoparticle of the present application, wherein: the photosensitizer loaded on the nanoparticle is obtained by mixing the tumor microenvironment-regulated nanoparticle and the photosensitizer-containing solution for a period of time; wherein the mass ratio of the nanoparticle to the photosensitizer is 400:1.25-80.

[0057] As a preferred scheme of the multifunctional nanoparticle of the present application, wherein: the reaction time is 24 h.

[0058] Another object of the present application is to provide the use of the tumor microenvironment-regulated nanoparticle as described above or the multifunctional nanoparticle as described above in the preparation of an antitumor drug.

[0059] Compared with the prior art, the present application has the following beneficial effects:

[0060] The application has the ability to respond to and adjust the tumor microenvironment (TME), and can realize the synergistic tumor treatment of photothermal and photodynamic under the guidance of photothermal and nuclear magnetic imaging under single NIR laser irradiation. The material has the advantages of simple preparation process, low cost and good biocompatibility, and can respond to and modify the tumor microenvironment. It can realize targeted, non-invasive and efficient killing of tumor cells, and has a broad clinical application prospect in tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0061] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor. Among them:

[0062] Figure 1 PMIC prepared by the present application Apt-ROX Characterization of PMIC / ICG; wherein (a) is the TEM image of PMI; (b) is the TEM image of PMIC; (c) is the SEM image of PMI; (d) is the SEM image of PMIC; (e) is the UV-Vis spectrum of PMI, PMIC, PMIC Apt-ROX , PMIC Apt -ROX / ICG, free ICG and Apt-ROX; (f) is the fluorescence spectrum of PMIC, Apt-ROX, PMIC Apt-ROX and PMIC Apt-ROX supernatant; (g) is the hydrodynamic diameter (DLS) and (h) Zeta potential of PMI, PMIC, PMIC Apt-ROX and PMIC Apt-ROX / ICG; (i) is the distribution of each element in PMIC. Scale: 200nm.

[0063] Figure 2 Photothermal performance characterization of PMIC in Example 3 of the present application; wherein (a) is the temperature change (ΔT) of PMIC (300μg / mL) solution under 808nm laser irradiation at different power densities (0.1, 0.3, 0.5, 0.8 and 1.0W / cm 2 ); (b) is the temperature change of PMIC solution at different concentrations (0, 12.5, 25, 50, 100, 200, 300μg / mL) under 808nm laser irradiation at a power density of 0.3W / cm 2 ); (c) is the photothermal stability test of PMIC solution after 6 cycles of laser on / off.

[0064] Figure 3 Test results for loading and controlled release of ICG in Example 4 of the present application; where (a) is the loading of PMIC Apt Loading of ICG in different concentrations of ICG solution. (b) is the release of ICG from PMIC Apt / ICG over time in different pH (5.0, 6.5 or 7.4) PBS.

[0065] Figure 4 Test results for incubation of PMIC Apt (0, 50, 100, 200, 500, and 1000 pg / mL) with GSH for 10 min or 3 h, followed by incubation with DTNB for 10 min. The ability of PMIC Apt to consume GSH was evaluated by the absorbance change of DTNB at 412 nm.

[0066] Figure 5 Results of O2 generation experiment in Example 6 of the present application: (a) Oxygen generation over time was detected using a dissolved oxygen meter for the following solutions: PBS + H2O2 (1 mM), PMIC (15 mg), and PMIC + H2O2 (1 mM). (b) 4T1 cells were incubated with PBS or PMIC Apt (300 pg / mL) for 4, 12, and 24 h, and the change in oxygen probe RDPP signal intensity was observed using confocal laser scanning microscopy (CLSM) to reflect the generation of oxygen in cells. Scale bar: 50 pm.

[0067] Figure 6 Results of in vitro ROS generation detection in Example 7 of the present application: The change in UV-visible absorption spectra of the solution over time under the following conditions: (a) 1 mM H2O2 + 808 nm (0.3 W / cm 2 ), (b) H2O2 (1 mM) + PMIC Apt / ICG (300 pg / mL), and (c) H2O2 (1 mM) + PMIC Apt / ICG (300 pg / mL) + 808 nm (0.3 W / cm 2 ); (d) is the CLSM fluorescence image showing the generation of ROS in 4T1 cells incubated with PBS, ICG, PMIC Apt , PMIC Apt / ICG, and treated with 808 nm 0.3 W / cm 2 (L) laser (ICG-L and PMIC Apt / ICG-L). Scale bar: 50 pm.

[0068] Figure 7 The results of tumor cell targeting assays for nanoparticles in Example 8 of this invention are shown; wherein, (a) is the result of 4T1 after PMIC. Apt / ICG or PMIC Ran (a) CLSM fluorescence image after 8 hours of ICG incubation; (b) after PMIC incubation. Apt / ICG or PMIC Ran After 8 hours of incubation with ICG, the cells were analyzed by flow cytometry for PMIC. Apt / ICG intake.

[0069] Figure 8 The results of the toxicity test of the nanoparticles in Example 9 of this invention are shown.

[0070] Figure 9 The results of cell viability determination after different nanoparticle treatments in Example 10 of this invention are shown.

[0071] Figure 10 This is a statistical histogram showing the percentages of necrotic, late apoptotic, early apoptotic, and viable 4T1 cells in each group after different treatments in Example 11 of this invention. The data are expressed as mean ± standard deviation (n=3).

[0072] Figure 11 The PMIC in Embodiment 12 of the present invention Apt / ICG photothermal (PTI) and magnetic resonance (MRI) dual-mode imaging; where (a) is 808 nm (1.2 W / cm²). 2 (a) Temperature change curves of the tumor site at different time points under laser irradiation; (b) Temperature change curves at 808 nm (1.2 W / cm²). 2 (c) Photothermal images of tumor sites at different time points under laser irradiation; (d) 1 / T2 and PMIC. Apt / The linear relationship of ICG concentration is shown in the inset, with different concentrations of PMIC. Apt / ICG corresponding T2-weighted MRI image; (d) is intravenous PMIC injection. Apt T2-weighted tumor MRI images at 0h (blue dot circle) and 24h (red dot circle) after ICG; (e) shows PMIC at different concentrations. Apt In vitro T1-weighted MRI images of / ICG (inset) and 1 / T1 versus PMIC Apt Relationship with ICG concentration; (f) Intratumoral injection of PMIC Apt / ICG images before (blue dot circle) and after (red dot circle).

[0073] Figure 12 This document presents the results of measuring the therapeutic effects of different treatment methods on 4T1 tumor-bearing mice in Example 13 of this invention. (a) shows photographs of tumors in 4T1 tumor-bearing mice on day 14 after different treatment methods; (b) shows the curves of relative changes in tumor volume over 14 days after treatment; (c) shows the average weight of tumors in each group on day 14 after treatment; and (d) shows the curves of relative changes in tumor volume over 14 days after treatment. Data are expressed as mean ± standard deviation (n = 4, *P < 0.05; **P < 0.01; ***P < 0.001); (e) shows representative images of H&E, TUNEL, Ki-67, CD31, and HIF-1α immunostaining in tumors of different treatment groups on day 14 after treatment. Scale bar: 100 μm.

[0074] Figure 13 This invention illustrates the effects of various treatments on the function of major organs in mice 14 days after treatment in Example 13 of this invention; wherein, (a) is the determination of AST in the serum of 4T1 tumor-bearing mice (n=3) under different treatments; (b) is the determination of ALT in the serum of 4T1 tumor-bearing mice (n=3) under different treatments; (c) is the determination of UREA in the serum of 4T1 tumor-bearing mice (n=3) under different treatments; and (d) is the determination of CPK in the serum of 4T1 tumor-bearing mice (n=3) under different treatments. Detailed Implementation

[0075] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0076] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0077] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0078] Unless otherwise specified, all raw materials used in the examples are commercially available.

[0079] Example 1

[0080] (1) Synthesis of PBMnIr (PMI) nanomaterial:

[0081] First, 20 mL of an aqueous solution containing citric acid (5 mM) and K3[Fe(CN)6] (1.0 mM) was added dropwise to 20 mL of an aqueous solution containing citric acid (5 mM), FeCl2(0.4 mM), MnCl2(0.3 mM), and IrCl3(0.3 mM). The solution turned bright blue after stirring at 60 °C for 30 min at 1200 rpm. Subsequently, the solution was centrifuged at 14000 rpm for 15 min, and then washed three times with deionized water, and the resulting nanoparticles (PMI) were collected. Finally, the washed sample was dried in a vacuum desiccator at 60 °C for 24 h.

[0082] (2) Synthesis of PMI@CaCO3(PMIC):

[0083] First, 7.5 mL of cyclohexane, 1.77 mL of Triton X-100, and 1.6 mL of 1-hexanol were mixed well at 600 rpm. Next, 2.5 mg of PMI was added under vigorous stirring, and then a 3 mM calcium chloride (800 μL) solution was added dropwise to the above solution. Then, a 292 mM sodium carbonate solution (80 μL) was added dropwise, and stirring was continued for 10 h. Finally, centrifugation was performed at 8500 rpm for 5 min, and 3 washes were performed using anhydrous ethanol and deionized water to obtain PMI@CaCO3(PMIC) nanoparticles.

[0084] (3) Synthesis of PMIC@Aptamer (PMIC Apt ):

[0085] First, 2.5 mg of PMIC was added to a phosphate buffer solution (10 mM PBS, pH 7.4) containing 1% bovine serum albumin (BSA) and incubated at 4°C for 10 hours. Then, the solution was centrifuged at 8500 rpm for 5 minutes to obtain PMIC@BSA. Subsequently, a solution of streptavidin with a concentration of 55 mg / ml (50 μL) was added to the PMIC@BSA solution (1 mL, 2.5 mg / ml) in a mixer and incubated at 4°C for 4 hours. The solution was centrifuged again at 8500 rpm for 5 minutes to remove the supernatant. Then, the streptavidin-loaded PMIC@BSA (1 mL, 2.5 mg / mL) was mixed with 25% glutaraldehyde (20 μL) in a PBS solution and stirred at room temperature for 2 hours. The aptamer was annealed in PBS containing 5 mM magnesium chloride using a PCR instrument (reaction conditions: 95°C for 5 min, slow cooling to 22°C, total time 10 min) to fold it into a three-dimensional structure. 100 μM (10 μL) of biotin-modified aptamer (Apt-Bio, Table 1) or the above-mentioned aptamer labeled with ROX fluorescent marker (ROX-Apt-Bio, Table 1) was mixed with PMIC@BSA in PBS and incubated at 4°C for 8 hours. Finally, three deionized water washes were performed to obtain PMIC Apt or PMIC Apt-ROX nanoparticles.

[0086] Table 1

[0087]

[0088] The present application first synthesized Prussian blue analog nanoparticles doped with manganese (Mn) and iridium (Ir), named PMI. First, the morphology of PMI was characterized using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) ( Figure 1 a, Figure 1 c), and the results showed that PMI maintained a regular cubic structure. At the same time, from the TEM image ( Figure 1 b) and the SEM image ( Figure 1 d), it can be seen that, compared with PMI, the PMIC particles coated with CaCO3 have a larger size and a rougher surface. In addition, after coating with CaCO3, the hydrodynamic diameter (DLS) of the particles increased from 328.7 ± 2.9 nm to 346.5 ± 3.8 nm ( Figure 1 g), and the Zeta potential decreased from -14.5 ± 0.7 mV to -28.3 ± 1.6 mV ( Figure 1 h), which indicated that the coating of CaCO3 was successful. The results of the major element analysis ( Figure 1 i) also proved the successful synthesis of PMIC.

[0089] This invention utilizes Rhodamine X (ROX)-modified TfR and EpCAM aptamers (ROX-Apt-Bio) on the surface of cancer cells to evaluate the modification of PMIC by the aptamers and their specific recognition of cancer cells. Figure 1 As shown in e, at 608nm, Apt-ROX and PMIC Apt-ROX Both exhibited strong fluorescence signals (ROX fluorescence). Simultaneously, the Apt-ROX-modified PMIC also showed an absorbance peak at 585 nm (the absorption peak of the ROX molecule), indicating that Apt-ROX was successfully attached to the PMIC. Figure 1 f). After Apt connection, the hydrodynamic diameter of the particles increased from 346.5 ± 3.8 nm to 444.2 ± 6.1 nm. Figure 1 g), the Zeta potential increased from -28.3±1.6mV to -16.2±0.1mV, further proving the success of the above connection. Figure 1 h).

[0090] Example 2

[0091] This Example 2 is based on the PMIC prepared in Example 1. Apt Based on nanoparticles, photosensitizers (indocyanine green, ICG) are further loaded to prepare PMICs. Apt / ICG.

[0092] Preparation of PMIC Apt The steps for nanoparticles are the same as in Example 1, and the following steps are performed: PMIC Apt A mixture of 0.4 mg / mL (500 μL) and 500 μL of ICG (10 μg / mL) solution was placed in a 1.5 mL tube, and PMIC was obtained after 12 hours. Apt / ICG.

[0093] The load of ICG makes PMIC Apt The nanoparticle size increased to 455.0 ± 1.6 nm. Figure 1 g), the Zeta potential decreased from -16.2±0.1mV to -14.8±0.1mV. Figure 2 h). The absorbance spectra of PMIC and ICG molecules partially overlap in the near-infrared (NIR) spectral region, which is beneficial for achieving synergistic photothermal and photodynamic therapy under near-NIR laser irradiation.

[0094] Example 3: Characterization of the photothermal properties of PMIC

[0095] To evaluate the photothermal properties of PMIC, 1 mL of PMIC (concentration 300 μg / mL) was added to a quartz test tube. Then, different laser densities (0.1, 0.3, 0.5, 0.8, and 1.0 W / cm²) were used.2 The above solution was irradiated with an 808 nm laser to investigate the effect of laser intensity on the solution temperature (an equal volume of deionized water was used as a negative control). Additionally, a power density of 0.3 W / cm² was used. 2 An 808 nm laser was used to irradiate 1 mL of PMIC at different concentrations (0, 12.5, 25, 50, 100, 200 or 300 μg / mL) to investigate the effect of nanomaterial concentration on solution temperature.

[0096] Experimental results are as follows Figure 2 As shown, when the concentration of PMIC is 300 μg / mL, its temperature change (ΔT) under 808 nm near-infrared light irradiation increases with increasing laser power density. Figure 2 a) When the laser power density is 1.0 W / cm² 2 At this temperature, ΔT can reach 44.2℃, demonstrating PMIC's ability as a highly efficient photothermal conversion agent. It is worth noting that ΔT after laser irradiation also exhibits a dependence on the PMIC concentration. Figure 2 b). The above results indicate that the temperature change of the PMIC solution after NIR laser irradiation is positively correlated with the 808 nm laser power density and the PMIC concentration.

[0097] To evaluate the photothermal stability of the above materials, an 808nm laser at 0.5W / cm² was used. 2 A 1 mL PMIC solution was irradiated six times at a power density. Each irradiation cycle consisted of a 10-minute heating period followed by a 20-minute natural cooling period, totaling 30 minutes per cycle. Temperature changes were monitored using a digital thermometer, and the photothermal conversion efficiency (η) of the PMIC was calculated. The experimental results are as follows: Figure 3 As shown in Figure c, after six laser on / off irradiation cycles, the temperature change of the PMIC-containing solution remained almost constant. By analyzing the heating and cooling curves, the photothermal conversion efficiency (η) of the PMIC was calculated to be 34.96%.

[0098] Example 4: Loading and release of photosensitizer

[0099] (1) ICG loading experiment:

[0100] First, standard curves for ICG were plotted by measuring the absorbance at 800 nm using ICG solutions of different concentrations, for subsequent quantitative analysis of ICG. Next, 500 μL and 0.4 mg / mL PMIC solutions were respectively... AptMix 500 μL of ICG solutions of different concentrations (0.625, 1.25, 2.5, 5, or 10 μg / mL) in a 1.5 mL tube. At specific time points, centrifuge the mixture at 8000 rpm for 5 minutes, and then measure the absorption spectrum of the ICG supernatant using a UV-Vis spectrometer. Afterward, return the supernatant to the PMIC. Apt In the ICG solution, continue to mix on a mixer. PMIC at different time points (1, 3, 6, 12, 24, and 36 hours). Apt The mass of ICG loaded in the nanoparticles will be calculated by subtracting the concentration of ICG in the supernatant from the initial ICG concentration in the solution.

[0101] like Figure 3 As shown in a, in PMIC Apt On the carrier, the ICG loading mass increased with increasing ICG concentration. When incubated with 10 μg / mL ICG, only 68% of the ICG was loaded onto the nanoparticles. Even when the ICG concentration was increased to 20 μg / mL, the ICG loading did not increase significantly. The maximum ICG loading reached 13.6 μg / mg (ICG / PMIC). Apt ).

[0102] (2) ICG release experiment: PMIC loaded with ICG (maximum loading capacity 13.6 μg / mg) was used. Apt ICG (200 μg) was placed in buffer solutions with different pH values ​​(5.0, 6.5, or 7.4) and incubated for 48 hours. The supernatant was then centrifuged for 5 minutes at specific time points (30 min, 1, 2, 3, 6, 9, 12, 24, 36, and 48 hours) and the absorbance was measured. The mass of ICG released into the supernatant was determined using an ICG standard curve, and the percentage of ICG released was calculated.

[0103] like Figure 4 As shown in b, the exchange rate of ICG from PMIC was compared under different pH conditions. Apt The release ratio in / ICG. It can be seen that, under pH 7.4 conditions, PMIC... Apt The proportion of ICG released by PMIC was 20.3%, while the release rate increased to 26.6% and 28.8% at pH 6.5 and 5.0, respectively. These results reveal the PMIC Apt pH response characteristics of / ICG to ICG release.

[0104] Example 5: Glutathione (GSH) Consumption Experiment

[0105] First, 25 mg of DTNB was dissolved in 10 mL of 0.1 M PBS to prepare a GSH detection probe. Then, freshly prepared PMICs at different concentrations (0, 50, 100, 200, 500, and 1000 μg / mL) were used. Apt After incubating with 100 μM GSH for 10 minutes or 3 hours, 10 μL of DTNB solution was added and incubated for another 10 minutes. Finally, the change in absorbance at 412 nm was measured by UV-Vis spectroscopy.

[0106] Using DTNB as an indicator molecule, GSH and different concentrations (0, 50, 100, 200, 500 or 1000 μg / mL) of PMIC were determined. Apt The changes in the 412nm absorption peak of DTNB after 10 minutes or 3 hours of exposure. Figure 5 The results show that, with PMIC Apt With increasing concentration and incubation time, the concentration of GSH continuously decreased. This result indicates that PMIC Apt It has good GSH consumption capacity.

[0107] Example 6: Oxygen Generation Experiment

[0108] 15 mg of PMIC nanoparticles were mixed with 30 mL of 1 mM H₂O₂ solution in a 50 mL centrifuge tube. Before testing, pre-dissolved O₂ in the solution was removed by continuous nitrogen purging. Changes in dissolved oxygen levels were measured using a dissolved oxygen meter under constant stirring conditions.

[0109] like Figure 5 As shown in Figure a, in a solution containing PMIC and H₂O₂, the oxygen concentration rapidly increased to 3.12 mg / L. In contrast, the oxygen produced in PMIC solutions without H₂O₂ and PMIC-free solutions containing H₂O₂ was negligible. This indicates that PMIC possesses catalase-like properties, capable of decomposing H₂O₂ into H₂O and O₂. In cancer treatment applications, PMIC can overcome tumor hypoxia by catalyzing the abundant H₂O₂ within cancer cells, thereby enhancing the efficacy of aerobic photodynamic therapy.

[0110] Detection of intracellular PMICs using oxygen probes (RDPP) Apt The process catalyzes the production of oxygen from hydrogen peroxide. First, cultured breast cancer cells (4T1 cells) were incubated with 5 μM RDPP for 4 hours. Then, PMIC was added to a final concentration of 300 μg / mL. Apt The cells were cultured and their fluorescence images were scanned using a confocal laser scanning microscope (CLSM) at different time points (4, 12, and 24 hours) under 488 nm excitation.

[0111] The generation of oxygen inside the cells was evaluated by using a fluorescence probe (RDPP) that is quenched by oxygen. As shown in Figure 6 b, the fluorescence induced by RDPP inside the cells was gradually quenched as the incubation time was prolonged, while the cells treated with PBS showed the strongest fluorescence signal. This indicates that PMIC Apt has a significant ability to generate oxygen inside the cells. Apt

[0112] Example 7: Detection of ROS generation in vitro

[0113] (1) Extracellular experiment: The generation of ROS was detected by measuring the absorbance change of ROS probe (DPBF) at 410 nm using a UV-Vis spectrophotometer. 0.5 mL of ethanol containing 40 μg / mL DPBF was mixed with 0.5 mL of PMIC Apt / ICG suspension (300 μg / mL) containing 1 mM H2O2. The above mixture solution was irradiated with a 0.3 W / cm 2 , 808 nm laser in a cuvette, and the absorbance of the solution was recorded every minute. DPBF+H2O2 with laser irradiation and DPBF+H2O2+PMIC Apt / ICG without laser irradiation were used as control groups.

[0114] The generation of ROS induced by PMIC Apt / ICG was evaluated by using DPBF, one of the ROS-related molecules. DPBF irreversibly reacts with the singlet oxygen generated by PDT, resulting in a decrease in the absorbance of DPBF at 410 nm. As shown in Figure 6 c, under the irradiation of 808 nm laser (0.3 W / cm 2 ), the absorbance of DPBF solution incubated with H2O2+PMIC Apt / ICG decreased significantly at about 410 nm as the time increased, indicating the generation of cytotoxic ROS in the solution. In contrast, as shown in Figure 6 a, no ROS was generated in the H2O2 solution with laser irradiation; and Figure 6 b, the PMIC Apt +H2O2 solution without laser irradiation.

[0115] (2) Intracellular experiment: The generation of ROS inside the cells was detected by using an intracellular ROS probe (DCFH-DA). The experimental procedure was as follows: 4T1 cells were seeded in a 96-well plate at a density of 1 x 10 4 cells per well, and incubated for 12 h. Then, PBS, ICG, PMIC Apt or PMIC​Apt After 12 h incubation with ICG, DCFH-DA was added to each well at a final concentration of 0.1 mM. After another 20 min incubation, the laser irradiation groups (ICG-L group and PMIC Apt / ICG-L group) were irradiated with 0.3 W / cm 2 , 808 nm laser for 10 min, and finally the intracellular fluorescence was recorded by CLSM (excitation wavelength: 488 nm).

[0116] The ability of PMIC Apt / ICG to induce intracellular ROS generation was evaluated using the fluorescent probe DCFH-DA. CLSM fluorescence imaging Figure 7 d) showed that PBS, ICG, PMIC Apt and PMIC Apt / ICG groups without laser irradiation showed weak fluorescence, indicating limited intracellular ROS generation. In contrast, cells treated with ICG-L showed relatively strong green fluorescence, indicating that ROS generation in normal PDT treatment groups was noticeable. Notably, cells in the PMIC Apt / ICG-L treatment group showed the highest fluorescence intensity. This phenomenon can be explained by the fact that PMIC Apt / ICG treatment leads to a decrease in intracellular GSH, and the oxygen generated by PMIC catalysis helps to alleviate the tumor hypoxic state, thereby enhancing the ability to generate ROS. Based on these findings, it can be speculated that PMIC has the potential to enhance the effectiveness of photodynamic therapy (PDT).

[0117] Comparative Example 1

[0118] This comparative example 1 is basically the same as Example 2, except that in the PMIC Apt synthesis step of step (3) in Example 1, the Apt-Bio modified nanoparticles were replaced with a random single-stranded DNA sequence (Random-Bio, Table 2); and further loaded with the photosensitizer ICG to obtain PMIC Ran / ICG.

[0119] Table 2

[0120]

[0121] Example 8: Tumor cell targeting assay of nanoparticles

[0122] 4T1 cells were seeded in a 96-well plate at a density of 1 x 10 4 cells / well and cultured in RPMI medium containing 10% FBS for 12 hours. Subsequently, PMIC Apt / ICG and PMIC Ran / ICG were added to the culture medium, respectively. / ICG. After incubation for 1, 2, 4, or 8 hours, the culture medium was removed and the cells were washed twice with PBS. Next, the cells were fluorescence-imagined using CLSM at an excitation wavelength of 561 nm and an emission wavelength of 785 nm. Additionally, 4T1 cells were cultured at 4 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density of 1 cell / well in 24-well plates and cultured for 12 hours. Then, PMIC was added to the cell culture medium. Apt / ICG or PMIC Ran / ICG. For the control group, an equal volume of PBS was added. Subsequently, after incubation for 1, 2, 4, and 8 hours, the cells were digested and collected by centrifugation. Finally, flow cytometry was used to detect the endocytosis efficiency of cells for nanoparticles modified with different nucleic acid sequences.

[0123] PMICs linked to random single-stranded DNA (PMICs) Ran Compared to / ICG, PMIC Apt / ICG-incubated 4T1 cells showed a stronger ICG fluorescence signal after 8 hours of culture ( Figure 7 a). Flow cytometry results ( Figure 8 b) Display, PMIC Apt 4T1 cells incubated with ICG showed a higher proportion of ICG-positive cells and a higher mean fluorescence intensity. After 8 hours of incubation, 73.6% of the cells showed red fluorescence, while PMIC... Ran In the / ICG group, the proportion of fluorescent cells was only 51.5%. This indicates that, compared with PMIC... Ran Compared to / ICG, PMIC modified with dual receptor-targeting aptamers... Apt / ICG has a higher affinity for 4T1 cells and a higher cell uptake efficiency, demonstrating a significant active targeting ability for cancer cells.

[0124] Example 9: Toxicity determination of nanoparticles

[0125] The toxicity of the nanoparticles was evaluated using the 4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT) assay. In short, 4T1 cells were used at a concentration of 1 × 10⁻⁶ cells. 4 The cells were seeded at a density of 100 cells / well in 96-well plates and incubated for 12 hours in RPMI medium containing 10% FBS. Subsequently, different concentrations (0, 12.5, 25, 50, 100, 150, 200, 300 μg / mL) of PMIC were added to specific wells. AptCells were cultured with ICG for 24 or 48 hours before measurement. The culture medium was then removed, and the cells were washed twice with PBS. Next, 10 μL of MTT solution (5 mg / mL) was added to each well, mixed with 90 μL of culture medium containing 10% FBS, and incubated at 37°C for 4 hours. Afterward, the culture medium was removed, cells were lysed with 100 μL of DMSO, and the absorbance at 570 nm was measured using a microplate reader.

[0126] PMIC was detected by MTT assay for cell viability. Apt The cytotoxicity of ICG did not decrease significantly with increasing concentration. Figure 9 This indicates that it has good biocompatibility.

[0127] Example 10: Cell viability assay after different treatments

[0128] Four T1 cells were seeded in each 96-well plate at a cell density of 1 × 10⁶ cells per well. 4 Cells were cultured overnight in 100 μL of RPMI medium containing 10% fetal bovine serum. Next, the following substances were added to each well: PBS (3 μL), PMIC... Apt (3μL, 10mg / mL), ICG (3μL, 0.5mg / mL), PMIC Apt / ICG (3μL, 10mg / mL). Incubate the cell plate at 37°C for 3 hours. Then use a power density of 0.5W / cm². 2 Cells irradiated with 808nm laser (PBS-L group, ICG-L group, PMIC group) Apt -L group, PMIC Apt (ICG-L group). After irradiation, the cells were washed twice with PBS and then incubated with fresh medium containing MTT (0.5 mg / mL) at 37°C for 4 hours. After incubation, the medium was carefully removed, and 100 μL LDMSO was added to lyse the cells. The absorbance was measured at 570 nm using a microplate reader.

[0129] Test results are as follows Figure 9 As shown, the synergistic treatment group (PMIC) was found to have Apt The therapeutic effect of ICG-L was significantly better than that of phototherapy alone (PTT and PDT groups). At 24 and 48 hours after treatment, the relative cell viability in the combined treatment groups was 1.97% and 1.68%, respectively, while the relative cell viability in the PTT and PDT groups was 16.73% and 22.02%, and 71.24% and 74.28%, respectively. Figure 10 ).

[0130] Example 11: Apoptosis Experiment

[0131] 4T1 cells were seeded into 24-well plates and cultured for 12 hours. Then, the following solutions were added to the wells: PBS and PMIC. Apt (10mg / mL), ICG (0.5mg / mL), PMIC Apt 12 μL of ICG (10 mg / mL) was added to each group, and incubated for another 3 hours. This was done in groups (PBS-L, ICG-L, PMIC). Apt -L group, PMIC Apt In the ICG-L group, use 0.3W / cm 2 After irradiation with an 808nm laser, the cells were cultured for 48 hours. After harvesting, the cells were washed twice with PBS and resuspended in 100μL binding buffer. The cells were then stained with PI (5μL) and Annexin V-FITC (5μL) and incubated in the dark for 10 minutes. Finally, flow cytometry was used to analyze the fluorescence of 20,000 cells collected from each group.

[0132] Histograms showing the percentages of necrotic, late apoptotic, early apoptotic, and viable 4T1 cells in each group after different treatments are shown below. Figure 10 As shown. Data are expressed as mean ± standard deviation (n=3). The percentage of surviving 4T1 cells in the co-treatment group was 4.17%, significantly lower than that in the PTT+L group (29.2%), PDT+L group (58.8%), and PBS control group (91.4%). Furthermore, the percentages of late and early apoptotic cells were also relatively higher in the co-treatment group. Figure 11 The above research results indicate that PMIC Apt / ICG is superior to single phototherapy in tumor treatment and has a more effective in vitro anti-cancer ability.

[0133] Example 12: Establishment and Imaging of an Animal Tumor Model

[0134] All animal experiments were conducted in accordance with the "Guidelines for Animal Welfare and Ethical Review" (GB / T 35892-2018) and the guidelines of Changchun Weishi Testing Technology Service Co., Ltd. (License No.: 20220930-01). Female BALB / c mice (6-7 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. 4T1 cells (2×10⁻⁶) were used. 6 Each mouse was subcutaneously injected with 100 μL of PBS into the right axilla until the tumor volume increased to 200 mm. 3 Or 100mm 3 At that time, the imaging and treatment experiments began separately.

[0135] (1) Photothermal imaging (PTI) of tumors: First, tumor-bearing mice were injected with PMIC via the tail vein. Apt / ICG. Subsequently, at different time points (0, 6, 12, 24, 48 hours), 1.2W / cm² was used. 2 The tumor site was irradiated with an 808nm laser for 10 minutes, and the real-time temperature and thermal images were recorded by a thermal imager.

[0136] Providing imaging-guided synergistic therapy in vivo is crucial for precision cancer treatment. Therefore, PMIC was evaluated. Apt / ICG imaging capabilities in 4T1 tumor-bearing BALB / c mice. Given PMIC Apt / ICG exhibits excellent photothermal properties in vitro, and we investigated its photothermal imaging capabilities in vivo using PMIC. Apt / ICG was administered intravenously, followed by the application of an 808nm laser (power density of 1.2W / cm²) to the tumor site at designed time intervals (0, 6, 12, 24, 48 hours). 2 Irradiation lasted for 10 minutes. During this process, the real-time temperature change curve was recorded using an infrared thermal imager. Figure 11 a) and photothermal images ( Figure 11 b). The study results indicate that intravenous PMIC administration... Apt The PTI signal detected 24 hours after ICG treatment was stronger than at other time points. Therefore, 24 hours is the optimal time for photothermal therapy. Furthermore, the temperature at the irradiated tumor site can reach up to 60°C. This temperature is considered sufficient to ablate the tumor.

[0137] (2) Magnetic resonance imaging (MRI): Using an MRI scanner to scan PMIC at different concentrations (0, 0.03125, 0.0625, 0.125, 0.25, 0.5 mg / mL) Apt / ICG solution was used to obtain NMR images, and relaxation times were recorded. A standard curve was then used to calculate the relaxation rate of the nanomaterial. In tumor MRI, small animal MRI imaging was used to obtain images of mice after injection of PMIC. Apt T1 and T2 weighted images before and 24 hours after ICG.

[0138] PMIC was evaluated using a 1.0T small animal MRI scanner. Apt / ICG imaging capability. Results are as follows: Figure 11 As shown in c and 11e, 1 / T1-weighted and 1 / T2-weighted MRI signals are compared with PMIC. Apt / ICG concentration was linear, and the MR images of aqueous solution also showed concentration-dependent imaging effect (inset, concentration gradually increased from left to right). PMIC Apt The longitudinal (rl) and transverse (r2) relaxivity of PMIC -1 s -1 These results indicated that PMIC Apt / ICG could be considered as a T1 and T2 weighted MRI contrast agent.

[0139] The MRI imaging ability of PMIC Apt / ICG in vivo was further evaluated. The in vivo experimental results showed that Figure 11 d), 24 hours after intravenous injection of PMIC Apt / ICG, the T2 weighted signal was significantly enhanced (red dot circle) compared with that before injection (0 hour, blue dot circle). This result confirmed that PMIC Apt / ICG could be highly accumulated in tumor sites and could be used for T2 weighted MRI imaging. In addition, T1 weighted MRI signal was evaluated by intratumoral injection. As shown in Figure 12 f, after injection of PMIC Apt / ICG, the T1 weighted signal at the tumor site (red dot circle) was also enhanced compared with that before injection (blue dot circle), indicating that this nano-platform could also be used as a T1 weighted MRI contrast agent. These research results showed that PMIC Apt / ICG had great application potential in the field of in vivo tumor imaging, and could be applied to image-guided precise cancer treatment such as MRI and PTI.

[0140] Example 13: In vivo synergistic phototherapy and long-term toxicity determination

[0141] When the tumor volume reached 100mm 2 , female BALB / c mice were randomly divided into 8 groups, 4 in each group. These groups were injected with PBS, ICG, PMIC Apt , PMIC Apt / ICG, PBS, ICG, PMIC Apt , PMIC Apt / ICG through the tail vein. After 24 hours, four groups of mice were irradiated with 1.2W / cm 2 density 808nm laser for 10 minutes each time (PBS-L group, ICG-L group, PMIC Apt -L group, PMIC Apt / ICG-L group). Subsequently, the tumor size and mouse body weight were measured every day. The formula for calculating the tumor volume was: tumor volume = length x width x width / 2.

[0142] On day 14 of intravenous injection, major organs and tumors of mice in each group were subjected to the following experiments: tumor tissues were subjected to terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), proliferating cell nuclear antigen (Ki67), platelet-endothelial cell adhesion molecule (CD31), and hypoxia-inducible factor-1a (HIF-1a) staining to evaluate the apoptosis, proliferation, angiogenesis, and hypoxia of tumor tissue cells, respectively. Hematoxylin and eosin (H&E) staining was also performed on the tumors and major organs of mice in each group to evaluate the PMIC Apt Effects on tumor growth and organ structure. Meanwhile, blood samples of all mice were obtained and centrifuged at 3000 rpm for 10 minutes to separate the serum of mice for the following blood biochemical analysis: glutamic-oxaloacetic transaminase (AST), glutamic-pyruvic transaminase (ALT), urea (UREA), and phosphocreatine kinase (CPK) levels. For the PBS group and PMIC Apt / ICG-L group, the concentrations of white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), platelets (PLT), and lymphocytes were also determined to evaluate the toxicity of treatment.

[0143] Representative images of tumor-bearing mice and their tumors in each group were taken Figure 12 a). By observing the changes in tumor volume Figure 12 b) and weight Figure 12 c), it was found that PMIC Apt -L, ICG-L, and PMIC Apt / ICG-L groups significantly inhibited tumor growth. Compared with single optical treatment groups (PMIC Apt -L and ICG-L), the tumor volume and weight of the synergistic treatment group (PMIC Apt / ICG-L) were significantly smaller, with some mice even having completely disappeared tumors. These results further confirmed that PTT&PDT synergistic treatment had a significant advantage in anticancer effect. Notably, the body weight of mice in the treatment groups was comparable to that of the control group Figure 12 d), indicating that different treatments had no significant effect on the growth of mice.

[0144] Pathological evaluation was performed on tumors in each group to determine the anti-tumor effect and biocompatibility of the nano-platform in more detail Figure 13 e). Using hematoxylin-eosin (H&E) staining, it was found that the degree of tumor damage in the synergistic treatment group was significantly higher than that in the PTT (PMIC Apt -L) and PDT (ICG-L) groups. Compared with the control group (PBS), PMIC Apt , ICG, PMIC AptThe damage during treatment was negligible in the / ICG and PBS-L groups. PMIC was observed by TUNEL staining of tumor tissue mediated by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling. Apt The ICG-L group showed the strongest green fluorescence in tumor tissue, indicating that more tumor cells were in an apoptotic state. Furthermore, immunostaining for proliferation-associated antigen (Ki-67) revealed that the synergistic treatment group significantly inhibited tumor cell proliferation. Immunostaining also showed a reduction in the vascular endothelial cell marker (CD-31), indicating that the synergistic treatment group maximally inhibited angiogenesis. Finally, staining for hypoxia-inducible factor 1α (HIF-1α), which is typically upregulated in hypoxic tumors, was used to assess oxygen production and hypoxia levels in vivo. The PDT group (ICG-L) showed predominantly red areas, indicating the most severe hypoxia in the tumor tissue. In contrast, the PMIC group… Apt The lowest HIF-1α expression was observed in the / ICG-L group, indicating that PMIC possesses catalase-like activity and can catalyze excess H2O2 in tumor tissue in vivo, thereby successfully alleviating tumor hypoxia. These findings suggest that PMIC... Apt / ICG has demonstrated outstanding capabilities in overcoming hypoxia and synergistic anti-tumor therapy.

[0145] Further evaluation of PMIC was conducted using serum biochemical analysis. Apt / ICG biotoxicity ( ​ PMIC injection Apt Following ICG administration, functional indicators related to liver function, including AST and ALT, renal function, including urea levels, and cardiac function, including creatine phosphokinase (CPK), remained within the reference ranges for healthy mice. These serum biochemical analysis results demonstrate that PMIC... Apt / ICG has good biocompatibility.

[0146] In the present application, manganese (Mn) and iridium (Ir) doped Prussian blue (PMI) is first synthesized as a photothermal nanocore material, and a calcium carbonate (CaCO3) protective layer (named: PMIC) that can be degraded in an acidic environment is coated on the surface of PMI to improve the biocompatibility and responsiveness to low pH in TME of the nanomaterial. In order to improve the tumor targeting of the nanomaterial, we connect a bifunctional aptamer (Apt) containing transferrin receptor (TfR) and epithelial cell adhesion molecule (EpCAM) expressed on tumor cells to the surface of PMIC, obtaining nanoparticles (PMIC Apt ) with tumor targeting recognition and enrichment ability. Subsequently, we load photosensitizer indocyanine green (ICG) onto PMIC Apt , forming the final tumor microenvironment responsive and regulatory nanodiagnosis and treatment integrated platform (PMIC Apt / ICG). Due to the overlapping absorption spectra of PMIC and ICG, simultaneous activation of photothermal and photodynamic therapy under single-wavelength near-infrared laser irradiation at 808 nm can be achieved. The incorporation of Mn enables the present nanoplatform to have the ability of magnetic resonance imaging (MRI) and catalyzing hydrogen peroxide to produce oxygen; the incorporation of Ir enables the platform to have the ability of GSH consumption through its variable valence. The ability of PMIC Apt / ICG to produce oxygen and consume GSH significantly improves the photodynamic therapy effect of the present nanoplatform. In summary, this nanodiagnosis and treatment platform has the ability to respond and regulate the tumor microenvironment, and can realize photothermal and photodynamic synergistic tumor therapy under the guidance of photothermal and magnetic resonance imaging under single-wavelength near-infrared laser irradiation, which has broad clinical application prospects in tumor diagnosis and treatment.

[0147] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application, which should be covered in the scope of the claims of the present application.

Claims

1. A method of preparing nanoparticles with tumor microenvironment modulation, characterized by: include, The core of synthesized nanoparticles: K3[Fe(CN)6] aqueous solution was added to a substrate containing Fe... 2+ Mn 2+ Ir 3+ The mixture was mixed in a solution, reacted in the presence of citric acid, centrifuged, washed, and dried to obtain Prussian blue analog nanoparticles (PMI). A protective layer is synthesized to cover the core: a sodium carbonate solution is added to a calcium chloride solution in which PMI is uniformly dispersed to react and obtain PMIC; The targeting agent was attached to the protective layer: PMIC was added to a PBS solution containing bovine serum albumin (BSA) to obtain PMIC@BSA; Then streptavidin was added to the PMIC@BSA and glutaraldehyde mixed solution for incubation, and the nucleic acid aptamer was annealed, folded into a three-dimensional structure using PBS containing magnesium chloride, and mixed with PMIC@BSA in PBS to prepare PMIC Apt ; The aptamer contains sequences targeting transferrin receptors and epithelial cell adhesion molecules expressed on tumor cells; the aptamer sequence targeting the transferrin receptors on tumor cells is GGCCGGTACCGCGC, and the aptamer sequence targeting the epithelial cell adhesion molecules on tumor cells is ACGGAGGTTGCGTCCGT.

2. The method for preparing Prussian blue analog nanoparticles as described in claim 1, characterized in that: The Prussian blue analog nanoparticles have a hydrodynamic diameter of 300 ~ 350 nm and a zeta potential of -10 ~ -20 mV.

3. The nanoparticle with tumor microenvironment modulation prepared by the method of claim 1 or 2. include, Core: Prussian blue analog nanoparticles (PMI); A protective layer, covering the surface of the core, wherein the protective layer is calcium carbonate; and, A targeting agent attached to a protective layer, wherein the targeting agent is a tumor cell targeting aptamer, the aptamer containing sequences targeting transferrin receptors and epithelial cell adhesion molecules expressed on tumor cells; the aptamer sequence targeting the transferrin receptors of tumor cells is GGCCGGTACCGCGC, and the aptamer sequence targeting the epithelial cell adhesion molecules of tumor cells is ACGGAGGTTGCGTCCGT.

4. The nanoparticles with tumor microenvironment modulation as described in claim 3, characterized in that: The protective layer covering the core surface increases the hydrodynamic diameter of the nanoparticles to 310 ~ 370 nm and reduces the Zeta potential to -20 ~ -40 mV.

5. The nanoparticles with tumor microenvironment modulation as described in claim 3, characterized in that: The targeting agent attached to the protective layer increases the hydrodynamic diameter of the nanoparticles to 330 ~ 450 nm and the Zeta potential to -10 ~ -20 mV.

6. A multifunctional nanoparticle, characterized by: include, Nanoparticles with tumor microenvironment modulation as described in any one of claims 3 to 5; and, Photosensitizer loaded on the nanoparticles.

7. The multifunctional nanoparticle of claim 6, wherein: The absorption spectrum of the photosensitizer overlaps with the absorption spectrum of the nanoparticles.

8. The multifunctional nanoparticle of claim 6 or 7, wherein: The multifunctional nanoparticles have a hydrodynamic diameter of 380 ~ 460 nm and a zeta potential of -10 ~ -20 mV.

9. The use of the nanoparticles with tumor microenvironment regulation as described in any one of claims 3 to 5, or the multifunctional nanoparticles as described in any one of claims 6 to 8, in the preparation of antitumor drugs.

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