CN115531542B - Composite material for inhibiting postoperative tumor recurrence, preparation method and application - Google Patents

Abstract

The invention discloses a composite material for inhibiting postoperative tumor recurrence, a preparation method and application thereof, and belongs to the technical field of medical nano materials. The preparation method disclosed by the invention comprises the following steps: adding a mixed solution of copper chloride and palladium (II) potassium chloride into the polyvinylpyrrolidone solution, stirring, adding ascorbic acid for ultrasonic treatment, and dialyzing to obtain the composite material for inhibiting postoperative tumor recurrence. Also discloses the application of the composite material in preparing medical materials for inhibiting postoperative tumor recurrence. The composite material prepared by the invention has adjustable enzyme-like activity, enhances oxidative stress in tumors, can directly kill glioma cells through CDT and PTT, can enhance immunity against glioma and indirectly kill glioma, and provides an effective photo-thermal immune treatment strategy of 'one stone two birds' for glioma patients.

Description

Composite material for inhibiting postoperative tumor recurrence, preparation method and application

Technical Field

The invention relates to the technical field of medical nano materials, in particular to a composite material for inhibiting postoperative tumor recurrence, a preparation method and application thereof.

Background

Glioblastoma (GBM) is the most common malignant primary brain tumor in clinic, with poor prognosis, high recurrence rate and mortality. The median overall survival of primary glioblastoma patients is only around 14.6 months. GBM treatment still relies primarily on standard surgical resection, radiation Therapy (RT) and/or Temozolomide (TMZ) chemotherapy, and complete elimination of tumors due to recurrence remains a key challenge. Therefore, the development of optimal therapies for recurrent GBM is critical for cancer patients. Immunotherapy is considered one of the most effective methods for treating cancer and has proven to have great potential in an increasing number of malignant tumors. With intensive research into the Central Nervous System (CNS), researchers have found microglial cells to play a leading role in low-grade gliomas. This glioma is characterized by a mutation in an enzyme called IDH. In high grade gliomas or glioblastomas associated with the normal IDH gene, more macrophages migrate from the blood circulation to the Tumor Microenvironment (TME), but far fewer T lymphocytes. Macrophages play a very important role in tumor development, progression, invasion and immune escape. Thus, altering TME, alleviating the immunosuppressive state, and increasing the entry of activated T lymphocytes into TME is very important.

Immunogenic Cell Death (ICD) is a cell death pathway and has become an important point in recent years for the therapeutic induction of anti-tumor immunity. Upon receiving ICD, the dying tumor cells release both damage-associated molecular patterns (DAMP) and tumor-associated antigens (TAA). Primary DAMP include, but are not limited to: calreticulin (CRT), adenosine Triphosphate (ATP), heat shock proteins (HSP 70 and HSP 90) and high mobility group box 1 (HMGB 1). During the initial phase of ICD induction, CRT translocates to the cell surface, heat shock proteins act as "eat me" signals, and extracellular released or secreted ATP acts as an effective "find me" signal during the mid-stage of cell death. Similarly, at the later stages of cell death, extracellular release of HMGB1 serves as an immunostimulatory "danger" signal. First, these DAMPs from ICDs recruit and activate the innate immune system by stimulating antigen presentation by Dendritic Cells (DCs), and then effect processing and presentation of T cell receptors. Second, these activated T cells subsequently infiltrate the tumor in large amounts and help eliminate residual cancer cells. At the same time, TAA released or exposed from dead cancer cells may further enhance antigen-specific T cell responses.

With the continued development of biomedical technology, various therapeutic modes of ICD inducers have been revealed, including but not limited to: conventional chemotherapy (anthracyclines, oxaliplatin, etc.), radiation therapy, and photodynamic therapy. This provides a means and challenge for glioma treatment. The standard treatment for gliomas is surgical excision followed by radiation and chemotherapy with TMZ (second generation oral alkylating agent, but without induction of ICD alone). For GBM, the treatment effect is poor, the side effect is large, and most of GBM still needs to be subjected to secondary operation. It is well known that tumors have unique microenvironments, such as mild acidity, high hydrogen peroxide (50-100 x 10 ^-6 M) and hypoxia. Hypoxia can induce tumor cell metastasis, ultimately leading to reduced efficacy. To solve this problem, H has been explored for tumor treatment ₂ O ₂ Responsive nanoenzymes. The nanoenzyme has Peroxidase (POD) and oxidase(OXD) -like Activity, H can be bound under TME ₂ O ₂ And O ₂ Is decomposed into Reactive Oxygen Species (ROS). In addition, the photo-thermal effect can enhance POD and OXD-like activity, thereby generating more ROS. This is a perfect combination of CDT and PTT. Notably, oxidative stress localized CDT/PTT may meet ROS-based oxidative stress standards. On the one hand, powerful ROS and PTT can kill tumor cells. On the other hand, oxidative stress based on ROS can further induce ICDs to produce secondary lesions on tumor cells. These findings open new approaches for designing artificial enzymes with ICD-related immunotherapeutic patterns.

The nano enzyme is an important component of the nano material and has the characteristics of multifunction, high stability, low cost and the like. These advantages compared to the natural enzymes make it superior to traditional therapies in practical applications. However, the nano-enzyme has high cost, and low-cost transition metal is generally selected to manufacture the bimetal composite material, so the nano-enzyme composite material is used for inhibiting postoperative glioma recurrence.

Disclosure of Invention

The invention aims to provide a composite material for inhibiting postoperative tumor recurrence, a preparation method and application thereof, so as to solve the problems in the prior art, the material has adjustable enzyme-like activity, can enhance oxidative stress in tumors and promote killing of the tumors, and can induce ICD, reverse immunosuppressive tumor microenvironment and enhance anti-tumor immune response, thereby eliminating residual glioma cells and preventing recurrence.

In order to achieve the above object, the present invention provides the following solutions:

the invention provides a preparation method of a composite material for inhibiting postoperative tumor recurrence, which comprises the following steps:

adding a mixed solution of copper chloride and palladium (II) potassium chloride into the polyvinylpyrrolidone solution, stirring, adding ascorbic acid for ultrasonic treatment, and dialyzing to obtain the composite material for inhibiting postoperative tumor recurrence.

Further, the preparation method specifically comprises the following steps: : dissolving 10-15mg of polyvinylpyrrolidone in 1-2mL of deionized water to obtain PVP solution, adding a mixed solution of copper chloride and palladium (II) potassium chloride into the PVP solution, stirring at 25-28 ℃ for 1-2min, adding 1-1.5mL of ascorbic acid, performing ultrasonic treatment for 15-20min, and dialyzing with deionized water through a dialysis bag with a molecular weight cut-off of 10-15kDa for 2-3 days to obtain the composite material for inhibiting postoperative tumor recurrence;

the mixed solution is a mixed solution of 0.3-0.5mL of 20mM copper chloride and 0.7-1mL of 20mM palladium (II) chloride potassium.

The invention also provides a composite material for inhibiting postoperative tumor recurrence obtained by the preparation method.

The invention also provides application of the composite material in preparing medical materials for inhibiting postoperative tumor recurrence.

Further, the composite material can absorb hemostatic fluid gelatin Surgiflo to play a role in inhibiting postoperative tumor recurrence.

Furthermore, the composite material is combined with gelatin Surgillo which can absorb hemostatic fluid to be injected into a patient, active oxygen is generated by assisting with chemo-dynamic therapy and photothermal therapy, and the oxidative stress of tumor cells is enhanced so as to kill the tumor cells and inhibit postoperative tumor recurrence.

Further, the composite material is combined with gelatin Surgiflo which can absorb hemostatic fluid to be injected into a patient, so that the death of immunogenic cells is induced, the micro-environment of immunosuppressive tumors is reversed, and the anti-tumor immune response is enhanced, so that the postoperative tumor recurrence is inhibited.

The invention also provides a medical material for inhibiting postoperative tumor recurrence, which comprises the composite material or a material obtained by combining the composite material with gelatin capable of absorbing hemostatic fluid.

Further, the tumor comprises a glioma.

The invention discloses the following technical effects:

the invention prepares a porous palladium-copper nanocluster PCN composite material which has adjustable enzyme-like activities (oxidase, peroxidase and catalase). Meanwhile, the ROS catalyzed by PCN enhances oxidative stress in tumors, ICD is further induced in photothermal therapy (PTT), the composite material is combined with gelatin Surgiflo which can absorb hemostatic fluid to form a hemostatic matrix system (Surgiflo@PCN), the Surgiflo@PCN directly kills glioma cells through chemical kinetics therapy (CDT) and PTT, ICD is induced, immunosuppressive Tumor Microenvironment (TME) is reversed, and anti-tumor immune response is enhanced, so that residual glioma cells are cleared and relapse is prevented, the Surgiflo@PCN can directly kill glioma cells through CDT and PTT, the anti-glioma immunity is enhanced, glioma is indirectly killed, and an effective photo-thermal immune therapy strategy of 'one-stone-two birds' is provided for GBM patients.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the structural and compositional features of a PCN; a is a TEM image and an EDS image of the PCN; b is the HRTEM image of PCN; c is STEM image and EDS mapping image of PCN; d-e are the X-ray diffraction and X-ray photoelectron spectrograms of PCN in sequence; f is the thermogravimetric analysis of PCN and PN;

FIG. 2 is a TEM image of PN;

FIG. 3 is a STEM and EDS map image of residual PVP on PCN;

FIG. 4 is a particle size distribution of PCN and PN; a is PCN; b is PN;

fig. 5 is a picture of PCN and PN stably dispersed in water;

FIG. 6 is a photograph of the dispersion of PCN in deionized water, PBS and simulated body fluids;

FIG. 7 shows degradation of PCN at various hydrogen peroxide concentrations;

FIG. 8 is a characterization of the OXD-like activity of PCN; a is TMB in N ₂ Air and oxygen are directly oxidized by PCN; b is the change of the OXD-like activity of PCN and PN at different temperatures; c is the absorbance spectrum and visible color change of TMB after incubation for 10 minutes in the presence of different concentrations of PCN, where 1:0 μg ·mL ^-1 ，2：7.5μg·mL ^-1 ，3：15μg·mL ^-1 ，4：22.5μg·mL ^-1 ，5：30μg·mL ^-1 ，6：45μg·mL ^-1 ，7：60μg·mL ^-1 ，8：75μg·mL ^-1 And M:75 mug.mL ^-1 1-8 contain TMB, M does not contain TMB; d is the change in OXD-like activity of PCN and PN at different pH;

FIG. 9 shows the results of enzyme kinetics of PCN, a being the results of measurement of the OXD-like activity of TMB catalytic oxidation of PCN and PN; b is PCN and PN are in H ₂ O ₂ Catalytic activity of (a); c is the comparison of catalase-like activities of PCN and PN; d is the temperature change of PCN solutions with different concentrations under 808nm laser irradiation; e is (1W cm) under 808nm laser irradiation only ^-2 ) Heating curves of PCN and deionized water; f is the photo-thermal stability study result of PCN under 808nm laser irradiation in the heating-cooling process of 5 cycles; g is the activity schematic diagram of PCN analogue enzyme; h is an image of PCN solutions of different concentrations; i is the linear relationship between signal intensity and PCN concentration;

FIG. 10 is POD-like activity profile of PCN; a is the absorption spectrum and visible color change of TMB after 10 minutes incubation; b is the catalytic activity of PCN in TMB substrate; c is the catalytic activity of PCN and PN at different temperatures; d is the catalytic activity of PCN and PN at different pH values;

FIG. 11 shows CAT-like activity profile of PCN; a is H ₂ O ₂ And PCN at different concentrations, wherein 1:0 mug.mL ^-1 ，2：3.75μg·mL ^-1 ，3：7.5μg·mL ^-1 ，4：15μg·mL ^-1 ，5：30μg·mL ^-1 ，6：45μg·mL ^-1 ，7：60μg·mL ^-1 Each bottle contains 100mM H ₂ O ₂ The red color of the bottle is marked as oxygen bubbles; b is the PCN and PN pair H at different times ₂ O ₂ The decomposition produces oxygen; c is the catalytic activity of PCN and PN at different pH values;

FIG. 12 is H ₂ O ₂ Results of reaction with PCN; a is H with different concentration ₂ O ₂ Is a light absorbance of (2); b is absorbance at 240nm as H ₂ O ₂ A calibration curve of concentration establishment; c is H ₂ O ₂ Absorbance detection results after reaction with PCN at different time points;

FIG. 13 shows detection of three enzyme-like activities of OXD, POD and CAT of PCN; a is PCN, feSO ₄ And CuSO ₄ Color change comparison photograph of the OXD-like activity of (b) is PCN, feSO ₄ And CuSO ₄ An OXD-like activity comparison statistical graph of (c); c is PCN, feSO ₄ And CuSO ₄ A color-changing contrast photograph of POD-like activity of (a); d is PCN, feSO ₄ And CuSO ₄ A comparative statistical plot of POD-like activity of (c); e is PCN, feCl ₃ And CuSO ₄ A comparative photograph of the color change of CAT-like activity; f is PCN, feCl ₃ And CuSO ₄ CAT sample activity comparison statistical plots of (c);

fig. 14 is a photo-thermal characteristic of PCN; a is a laser irradiation at 808nm (1.0 W.cm) ^-2 ) Temperature variation of PCN and PN solutions of the same concentration; b is the measurement of the power density (1.0, 0.8, 0.5 w.cm) under 808nm laser irradiation ^-2 ) Temperature change of the PCN solution;

FIG. 15 is the ability of PCN to cross the Blood Brain Barrier (BBB) in vitro; a is a schematic diagram of BBB permeation model using a Transwell system; b is PN, PCN and PCN+NIR (806 nm,0.1W cm) ^-1 ) BBB permeability comparison of (b); c is the result of quantitative analysis of cell uptake of the PCN by U87 cells by a flow cytometer; d is O in U87 cells under low oxygen environment ₂ A generated fluorescent image; e is the localization of PCN in U87 cells; e is a TEM image of PCN in U87 cells after 6 hours incubation; g is intracellular ROS levels in various post-treatment U87 cells; h is a confocal image of U87 cells stained with calcein AM (green, living cells) and propidium iodide (red, dead cells) after different treatments in a simulated TME; i is the relative survival rate of U87 cells after different treatments; group 1, control, group 2, NIR only, group 3, PN, group 4, PCN and group 5, PCN+NIR; PCN or PN 100 g.mL ^-1 Near infrared laser, 808nm, 1w.cm ^-2 ，5min；

FIG. 16 shows the intracellular O production of different groups ₂ Fluorescence intensity comparison of (2);

FIG. 17 shows the results of quantitative analysis of reactive oxygen species production in different groups of cells;

FIG. 18 is a photograph showing the result of monitoring PCN uptake by intracranial tumors using Photoacoustic (PA) imaging; a is in vivo PA imaging of a U87 tumor-bearing mouse after intravenous injection of PCN; b is a time dependent tumor PA signal based on PA imaging data in a; c is the fluorescence image detected in each group of treated tumors, nuclei and hypoxic areas are shown with DAPI (blue) and anti-HIF-1 a antibodies (green); d is surgical craniectomy performed on U87 tumor-bearing mice after tumor inoculation, black circles represent surgical bone windows; e is U87 tumor irradiated by 808nm laser (1.0W cm) ^-2 5 min) temperature profile; f is T2W-MRI imaging of U87 tumor-bearing mice following different treatments (including control group, PN, PCN, and pcn+nir); yellow circles represent tumors; g is the average tumor growth curve of the mice in j; h, i is the survival curve and weight change of U87 tumor-bearing mice treated with each formulation; j is the 3D reconstruction of intracranial tumor size; k is TUNEL staining of glioma apoptosis from different treatment groups;

FIG. 19 a is a representative photograph of PCNs of various geometries, surgiflo and Surgiflo@PCNs; b is a representative scanning electron microscope image of Surgiflo and surgiflo@pcn; the scale bar is 100 mu m; c-d is the quantitative analysis of GL261 cell uptake of Surgiflo@Cy3-PCN by flow cytometry using a Transwell system; e is STEM image and EDS mapping image of Surgillo@PCN, and the scale is 10 μm; f is in vitro CRT exposure of different post-treatment GL261 cells as determined by flow cytometry in a simulated TME; g is the quantitative percentage of CRT exposure in the different groups; h is extracellular ATP secretion of GL261 cells assessed using ATP kit; i is the HMGB1 drug release profile detected using an enzyme linked immunosorbent assay (ELISAkit); j is flow cytometry analysis of DC maturation following stimulation with pre-treated GL261 cells for each formulation; k is the frequency of DC activation in the different groups;

FIG. 20 a is the relative viability of normal cells (HUVECs) with different concentrations of PCN; b is in the simulated TME with or without laser irradiation (806 nm, 0.8w.cm ^-2 ) In the case of (1), GL261 cells and PCN (100. Mu.g.mL) ^-1 ) Relative survival rate after incubation; c is intracellular ROS levels, PCN or in GL261 cells treated in each groupPN:100g·mL ^-1 Near infrared laser, 808nm, 0.8w.cm ^-2 ，5min；

FIG. 21 is a graph showing the results of a Surgiflo@PCN post-operative recurrence experiment for glioma inhibition; a is a schematic diagram of experimental design; b1 is an image of a tumor (purple arrow) formed in the brain of the mice 14 days after implantation; b2, tumor is resected from brain, GBM cavity (black arrow), bone window (red arrow) microsurgery; b3, surgiflo@PCN is in the cavity; b4, cutting off the tumor; c-d are the temperature change curves of the operation of irradiating the excision cavity and the tumor by using laser with different laser power densities at 808 nm; e, g is representative bioluminescence analysis image (e) and quantified signal intensity (g) (n=3 biologically independent animals per group); f is the survival curve of mice carrying GL261 treated differently (n=6 mice per group); h is representative HE and Tunel stained images of brain tissue from GL 261-carrying mice showing tumor resected cavities, blue circles representing tumor cavities; yellow circles indicate infiltrating residual tumors; the red circle represents residual Surgiflo;

FIG. 22 is an in vivo mechanism by which brain tissue enhances anti-tumor immune responses; a is a schematic diagram of experimental design; b, d is representative flow cytometry analysis of T cell infiltration in brain tissue with glioma after excision on cd3+ cells (b) and quantification results in different groups (d); c, e is representative flow cytometry analysis (c) and relative quantification (e) of cd4+foxp3+ T cells gated on cd3+cd4+ cells; f is the ratio of cd8+ cytotoxic T cells to cd4+ foxp3+ Tregs; g-i is the secretion level of IL-6, TNF- α and IFN- γ in brain tissue after each treatment;

FIG. 23 is a flow cytometry analysis of CD4+ T cell quantification in various groups of brain tissue;

FIG. 24 shows the hemolysis rate of PCN nanoenzymes at different concentrations;

FIG. 25 shows the results of blood routine examination of mice after intravenous injection of PCN nanoenzyme and PBS, respectively; a-h are AST, ALT, BUN, WBC, RBC, HCT, HGB, PLT in sequence;

FIG. 26 shows the results of H & E staining of major organs of PCN-treated mice.

Detailed Description

Preparation of porous palladium-copper nanocluster (PCN) composite material

1.1 preparation method

10mg of polyvinylpyrrolidone (PVP) was dissolved in 1mL of deionized water to obtain a PVP solution, and then 20mM copper chloride (CuCl) was added to the PVP solution ₂ ·6H ₂ O) (0.3 mL) and 20mM Palladium (II) Potassium chloride (K) ₂ PdCl ₄ ) (0.7 mL) and stirring at room temperature (25 ℃) for 1min, then adding 1mL of ascorbic acid into the reaction system rapidly, finally, carrying out ultrasonic treatment for 15min, and dialyzing with deionized water for 3 days, wherein the molecular weight cut-off of a dialysis bag is 10kDa, and the obtained product is the PCN composite material.

According to the method, deionized water is used for replacing copper chloride, and pure palladium nano Particles (PN) are obtained.

Material properties of 2 PCN

2.1 Experimental methods

Transmission Electron Microscopy (TEM), high Resolution TEM (HRTEM), scanning Transmission Electron Microscopy (STEM), energy dispersive X-ray (EDX) mapping of the catalyst was performed on a JEM-2100 electron microscope (JEOL, japan) with an acceleration voltage of 200 kV. Field emission Scanning Electron Microscope (SEM) observations were made on a reglus 8230 microscope (japan). X-ray diffraction (XRD) analysis was carried out on a D/MAX 2550VB/PC diffractometer (Japanese university of technology). X-ray photoelectron spectroscopy (XPS) was performed on the rbid-upgraded PHIe5000C ESCA system (perkineler, usa). Fourier transform Infrared Spectroscopy (FTIR) was recorded in the range of 400-4000cm-1 on a VERTEX70 spectrometer (Brookfield Germany). Thermogravimetric analysis (TGA) was performed using a DISCOVERY TGA550 system (TA Instruments, USA) at a temperature range of 30-900 ℃ under an air atmosphere. The surface area was calculated from the adsorption data using the Langmuir and Brunauer-Emmett-Teller (BET) methods. Raman spectra were captured on a Renishaw (Renishaw, uk) confocal microscope raman spectrometer.

2.2 experimental results

PCN is Cu assisted by ultrasound in the presence of polyvinylpyrrolidone (PVP) and ascorbic acid ²⁺ And Pd (Pd) ²⁺ Is easily synthesized by mild reduction reaction, which is a 30nm-40nm small grain nano particle assembly, as shown in the figurePlot a of 1 is a Transmission Electron Microscope (TEM) image and an energy dispersive X-ray spectroscopy (EDS) image of PCN, mesopores can be observed, and no voids in PN (fig. 2). Meanwhile, the apparent lattice fringes in the High Resolution Transmission Electron Microscope (HRTEM) image verify that the crystalline nanoparticles, especially the lattice distance of 0.22nm, suggests a typical (111) crystal plane of PCN alloy (b-plot of fig. 1). Accordingly, scanning Transmission Electron Microscope (STEM) images and EDS mapping images showed a uniform distribution of copper elements in the Pd matrix confirming the PCN alloy, which well assembled into uniform porous nanoparticles (fig. 1, c). This alloying effect can also be revealed by X-ray diffraction (XRD) patterns and X-ray photoelectron spectroscopy (XPS) spectra, where a much wider diffraction of PCN than PN means small grains with rich defects, and the negative shift of the Pd 3d binding energy of PCN compared to PN suggests down-regulation of the d-band center by Cu doped Pd (see d-e of fig. 1), and furthermore, additional Cu XPS signals, cu2p 3/2 and Cu2p 1/2, appear in PCN beyond PN, which is very consistent with EDS mapping results, consolidating alloys formed by Pd and Cu. Thus, the porous and defective structure with down-regulated d-band centers promotes catalytic applications of PCNs because they have high activity. During PCN formation, PVP stabilizer was found to remain on the PCN as shown by the C and N signals in STEM and EDS spectra (fig. 3), wherein thermogravimetric analysis (TG) showed that the relative content of PVP reached even about 70 wt% (f of fig. 1). This special polymer decoration ensures excellent water dispersibility of the nanoparticles, which have a Dynamic Light Scattering (DLS) particle size only slightly larger than the TEM-measured particle size (a-b of fig. 4) due to polymer-mediated hydrophilization. Thereafter, the synthesized nanoparticles could be well dispersed in various stimulated physiological media without precipitation in water even when centrifuged at 12000rpm for 12 minutes (fig. 5). Thus, the unique structure and properties of PCN offer promise for its biological applications. In addition, the prepared PCN was well dispersed in various solvents such as Simulated Body Fluid (SBF), PBS, and deionized water, indicating that PCN had good stability in these media (fig. 6). Importantly, it is rarely decomposed even at high hydrogen peroxide concentrations (fig. 7).

Oxd, POD and CAT-like Activity and kinetic assay of 3PCN

3.1 Experimental methods

3.1.1 detection of OXD-like Activity

PCN oxidized 3,3', 5' -Tetramethylbenzidine (TMB) in NaAc buffer (0.1M, pH 5.0) to produce a blue signal. Kinetic measurements of the PCN oxidase reaction were assessed at 652nm by a multi-function microplate reader (Thermo Scientific, varioskan Lux, usa). Typically, 10. Mu.L of PCN (final concentration 7.5. Mu.g.mL ^-1 ) To 200. Mu.L of NaAc buffer containing 10. Mu.L of TMB (final concentration 0.416 mM) was added to show a chromogenic reaction. Steady state kinetic analysis in 0.2mL NaAc buffer, 10. Mu.L PCN solution (final concentration 7.5. Mu.g.mL ^-1 ) And TMB. By adding different amounts (1, 2, 4, 6, 8, 10, 12.5, 20. Mu.L) of TMB solution (2 mg. Mu.L) ^-1 In DMSO), kinetic analysis of PCN was performed with TMB as substrate. Absorbance of all reactions was monitored at different reaction times and the mie constant was calculated from the mie-gate Teng Baohe curve by GraphPad Prism 7.0 (GraphPad software). The temperature dependence of the OXD-like activity of PCN was detected at different temperatures from 25 ℃ to 55 ℃ and the pH dependence was detected in different buffer solutions with pH values from 2 to 9. For comparison, the OXD-like activity of PN was also measured.

3.1.2 detection of POD-like Activity

H in NaAc buffer (0.1M, pH 5.0) using TMB as substrate ₂ O ₂ In the presence, POD-like activity assay of PCN was performed at 652nm by a multi-functional microplate reader (Thermo Scientific, varioskan Lux, USA). In a typical experiment, 10. Mu.L of PCN (final concentration 7.5. Mu.g.mL ^-1 ) Into 200. Mu.L of NaAc buffer containing 10. Mu.L of TMB (final concentration 0.416 mM) and 10. Mu. L H ₂ O ₂ (final concentration 50 mM) to show a chromogenic reaction. In 0.2mL of NaAc reaction buffer and 10. Mu.L of PCN solution (final concentration: 7.5. Mu.g.mL) ^-1 )、H ₂ O ₂ And steady state kinetic analysis was performed in TMB. By adding 10. Mu.L TMB (2 mg. ML) ^-1 In DMSO) and varying amounts (1, 2, 4, 6, 8, 10, 12, 14, 16, 20, 30, 40 μl) of H ₂ O ₂ Solution (1M), in H ₂ O ₂ Kinetic assays of PCN were performed for the substrate. By adding 10μL 1M H ₂ O ₂ And different amounts (1, 2, 4, 6, 8, 10, 12.5, 15, 17.5, 20. Mu.L) of TMB solution (2 mg. Multidot.mL) ^-1 DMSO) was used for PCN kinetics assays with TMB as substrate. All reactions were monitored by measuring absorbance at different reaction times and the mie constant was calculated from the mie-gate Teng Baohe curve by GraphPad Prism 7.0 (GraphPad software). The temperature dependence of the POD-like activity of PCN was detected at different temperatures from 25 ℃ to 55 ℃ and the pH dependence was detected in different buffer solutions with pH values from 2 to 9. For comparison, the POD activity of PN was also measured.

3.1.3CAT-like Activity

O was measured by using a specific oxygen electrode on a multiparameter analyzer (JPSJ-606L, leici China) ₂ The PCN was produced and assayed for CAT-like activity. Typically, 25. Mu.L of PCN (final concentration 7.5. Mu.g.mL ^-1 ) Adding a solution containing 100. Mu.L of 0.1. 0.1M H ₂ O ₂ In 1mL PBS buffer (0.1M pH 7.0). Measuring O at different time points ₂ Is generated. By adding different amounts (50, 100, 200, 300, 400, 500, 750 and 1000. Mu.L) of H ₂ O ₂ Solution (0.1M), in H ₂ O ₂ Kinetic analysis of PCN was performed for the substrate. Mirabilin constants were calculated from Mirabilin-gate Teng Baohe curves by GraphPad Prism 7.0 (GraphPad software). The pH dependence was detected in different buffer solutions with pH values of 3 to 9. For comparison, CAT-like activity of PN was also measured.

To detect H ₂ O ₂ Consumption to H ₂ O ₂ To the (10 mM) solution was added 10. Mu.L of PCN (final concentration 7.5. Mu.g.mL) ^-1 ). Five minutes later, the solution was centrifuged and the remaining H was recorded by a UV-vis spectrophotometer (Shimadzu UV-2600) at 240nm ₂ O ₂ Is a solid phase, and is a liquid phase. Evaluation of H against calibration Curve ₂ O ₂ By recording H at 240nm at various known concentrations ₂ O ₂ (1-50 mM).

3.2 experimental results

Nanoenzyme-inducible O having OXD-like activity or POD-like activity ₂ Or H ₂ O ₂ Decomposition generating survivalSex oxygen, thereby resulting in catalytic oxidation of 3,3', 5' -Tetramethylbenzidine (TMB) to form a blue product (oxTMB) with an absorbance of 652 nm. The present invention studied the Oxidase (OXD) like activity of PCN under specific pH and temperature conditions, and as a result showed that PCN and PN have significant pH and temperature dependence (b, d of fig. 8). The optimum pH and temperature ranges are 4.5 to 5.5 and 35 to 45℃respectively, and the catalytic activity of PN is lower under the same conditions. It was also found that the Vmax value of TMB catalyzed oxidized PCN was increased 1.6 times relative to PN (a of fig. 9), and these results confirm that PCN has higher catalytic activity than PN. Pure O was also evaluated ₂ Air and N ₂ The OXD-like activity in (a). It was found that pure O compared with air ₂ The enzymatic reaction in (a) is significantly increased, N ₂ The enzyme-catalyzed reaction in the atmosphere is significantly reduced (fig. 8 a). The role of oxygen in the OXD-like activity was further confirmed. FIG. 8 c is the absorbance spectrum and visible color change (0.1 MNAAc buffer, pH 5.0) of TMB (0.416 mM) after incubation for 10 min in the presence of different concentrations of PCN, wherein 1:0 mug.mL ^-1 ，2：7.5μg·mL ^-1 ，3：15μg·mL ^-1 ，4：22.5μg·mL ^-1 ，5：30μg·mL ^-1 ，6：45μg·mL ^-1 ，7：60μg·mL ^-1 ，8：75μg·mL ^-1 And M:75 mug.mL ^-1 1-8 contain TMB, M does not contain TMB; data are shown as mean ± standard deviation (n=3 biologically independent experiments).

TMB at H ₂ O ₂ POD-like activity of PCN and PN was studied in the presence of H ₂ O ₂ The decomposition generates ROS (e.g., OH). PCN shows higher POD-like activity compared to PN due to the presence of abundant active catalytic sites (a of fig. 10). Catalytic activity is also related to pH and temperature (c, d of fig. 10). The maximum pH and temperature of the catalytic activity of PCN and PN are pH 5.0 and 40℃respectively. By H ₂ O ₂ And TMB as substrates, a typical Mies kinetic equation was established. PCN and PN showed significant catalytic activity in both substrates (b of fig. 9 and b of fig. 10). TMB and H compared to PN ₂ O ₂ The Vmax value of the upper PCN was increased by 4.6-fold and 6.1-fold, respectively. It is clear that PCN has high catalase-like activity. Thus, all these results are advancedOne step has shown that PCN can be used as an effective enzyme-like activity for catalyzing H ₂ O ₂ And generates active oxygen toxic to cancer cells.

To study H ₂ O ₂ Decomposition into O ₂ And CAT-like Activity of PCN in Water, O in solution was measured by dissolved oxygen meter ₂ Concentration. We found H ₂ O ₂ O of solution ₂ The concentration increased rapidly with increasing PCN concentration, with the optimum pH value in the neutral range (pH 7.0) (fig. 11 a, c). PN was further studied and found to be less active than PCN (b of FIG. 11). As shown in FIG. 9 c, PCN decomposes H ₂ O ₂ The milbewill kinetics were followed. In the comparison between PCN and PN, the Vmax value of PCN is slightly higher than PN, however, the Km value is much lower than PN, which means for H ₂ O ₂ Is a strong affinity and high catalase-like activity. Furthermore, by measuring H at 240nm ₂ O ₂ Monitoring H in PCN catalyzed reactions ₂ O ₂ Concentration over time, and a standard curve was established (a, b of fig. 12). We observed H ₂ O ₂ The concentration continuously decreased within 5 minutes after the reaction, which was comparable to O ₂ The generated trends are consistent (c of fig. 12 and b of fig. 11). H ₂ O ₂ Consumption and O ₂ The close relationship between the generation can improve the local hypoxia condition of the tumor.

Taken together, these results indicate that PCN has three enzyme-like activities under physiological conditions: OXD, POD, and CAT. Further comparing classical Fenton reaction ions, PCN is found to have higher catalytic activity under the same molar concentration environment, which not only embodies the advantages of nano materials, but also verifies that PCN has higher catalytic performance (a-f of FIG. 13). The results show that PCN is more reactive than PN, which demonstrates that bimetallic PCN has higher catalytic activity than monometal PN. PCN can be used as a peroxisome mimetic, whereby cellular ROS can be regulated.

Photothermal Effect evaluation of 4 PCN

4.1 evaluation method

First, PCN (0, 12.5, 25, 50, 100. Mu.g.multidot.mL) was prepared at various concentrations ^-1 ) By combining 808nm laser (China Length)Spring laser photoelectric technology Co., ltd.) was irradiated with 1.0 W.multidot.cm in a glass bottle (200. Mu.L) ^-2 10 minutes. During irradiation, the temperature change of the solution was monitored by a thermocouple (provided by the smart drug delivery emphasis laboratory at the university of double denier). Next, the concentration was set to 100. Mu.g.mL ^-1 For further photothermal performance characterization. At different power densities (0.5W cm) with 808nm laser ^-2 、0.8W·cm ^-3 And 1.0 W.cm ^-4 ) PCN was irradiated for 10min. The temperature change was monitored during the irradiation. Under 808nm laser irradiation, the PCN solution (100. Mu.g.mL ^-1 1 mL) were measured for five cycles of real-time temperature. Each cycle included 5 minutes of irradiation followed by a 5 minute cooling period.

4.2 experimental results

Significant concentration-dependent and laser power-dependent photothermal effects of PCN (d of fig. 9 and b of fig. 14) were observed within 5 minutes, and infrared thermal images showed that at 100 μg·ml ^-1 The temperature was increased from 24.2 ℃ to 58.4 ℃ without significant temperature change being observed in deionized water (e of fig. 9). Then, the photo-thermal stability of PCN was high after five on/off laser cycles under continuous laser irradiation (f of fig. 9), and g of fig. 9 is a schematic diagram of the activity of PCN-like enzyme. In addition, the same concentration (100. Mu.g.mL) ^-1 ) The photothermal properties (. DELTA.. Apprxeq.27.1 ℃) of PN (1.0 W.multidot.cm) are slightly higher than those of PN (DELTA.. Apprxeq.24.2 ℃) ^-2 5 minutes) (a of fig. 14). In addition, PTT-binding reactive oxygen species production has proven to be an effective strategy for killing cancer cells. In addition, PCN may generate strong acoustic signals. As shown in h and i of FIG. 9, PCN in aqueous solution is linearly related to the PA signal (R ² =0.978), data are shown as mean ± standard deviation (n=3 biologically independent experiments), concentration increase ranging from 0 to 1000 μg mL ^-1 . Thus, PCN can be used as a PA contrast agent to guide the irradiation position and time, and PCN contrast PA imaging offers great potential for tumor visualization.

5 Effect of proliferating cell Nuclear antigen on in vitro tumor cells

5.1 Experimental design

5.1.1 cell culture: u87, GL261 and Luci+GL261 cells were purchased from the aboveXinzhou biotechnology limited of sea bridge. The cell lines were cultured in complete Dulbecco's Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 1% penicillin G sodium and 1% streptomycin sulfate. Cells were exposed to 95% air and 5% CO at 37℃ ₂ Incubate in humidified atmosphere and subculture was performed by addition of 0.25% trypsin and complete DMEM.

5.1.2 cellular uptake and subcellular distribution

To observe cellular uptake and subcellular distribution of PCN in U87 cells, PCN was labeled with Cy 3. Cy3 was slowly added to 1mL of PCN suspension, and the mixture was then magnetically stirred at room temperature in the dark for 48 hours. After dialysis (Merck Millipore, USA) of the solution against PBS (pH 7.4) to remove unbound Cy3, cy 3-labeled PCN (Cy 3-PCN) was obtained.

Digesting the U87 cells to obtain a cell suspension, and inoculating it into each well 10 ⁵ In 6-well plates of individual cells. After 24 hours of incubation, the medium was changed and then 100. Mu.g.mL per well ^-1 Cy3-PCN was added at the concentration of (C). After 0, 1, 2, 4, 6 hours, the cells were washed twice and then analyzed by flow cytometry (BD FACS Calibur, usa).

To track subcellular distribution of PCN, U87 cells were incubated with Cy3 PCN for 6h. To show lysosomes, cells were stained with Lyso Tracker Green (Beyotime Biotechnology, china) and Hoechst, respectively, for 30 min at RT. Cells were washed with PBS prior to fluorescent microscopy (zeiss, germany).

5.1.3 in vitro cytotoxicity assay

PCN biocompatibility experiments were performed on HUVEC. Cells were plated in 96-well plates at 0.8X10 per well ⁴ Density culture of individual cells and at 37℃at 5% CO ₂ Incubated in 100. Mu.L of DMEM medium for 24 hours. Continuous concentrations (0, 25, 50, 100, 200. Mu.g.multidot.mL) ^-1 ) PCN of (C) is added to the above medium. After 24 hours incubation, 10. Mu.L of CCK-8 was added to each well. Plates were incubated at 37 ℃ for 2 hours and then assayed in a multimode microplate reader (BioTek, synergy2, usa) at 450nm optical density.

To test cytotoxicityThe assay was performed with U87 cell suspensions at 0.8X10 per well ⁴ The density of individual cells (in 100 μl DMEM medium) was seeded in 96-well plates. Subsequently, PCN (0, 12.5, 25, 50, 100. Mu.g.multidot.mL ^-1 ) And H ₂ O ₂ (100. Mu.M) was added to the medium (pH 6.5). After 6 hours, 100. Mu.g/mL was used ^-1 PCN-incubated U87 cells were irradiated with 808nm laser (1.0W cm ^-2 ) Irradiation was carried out for 5 minutes. The cells were then further incubated for 24 hours. Finally, cell viability was assessed by CCK-8 kit assay according to the manufacturer's instructions.

For live/dead staining, U87 cells were stained at 2X 10 per well ⁴ The density of individual cells was seeded in 24-well plates. U87 cells were compared with control, NIR (806 nm, 1.0W.cm) ^-2 5 minutes), PN (100. Mu.g.mL) ^-1 )、PCN(100μM mL ^-1 ) And PCN+NIR H at pH 6.5 ₂ O ₂ (100. Mu.M) co-incubation. The cells were then further incubated for 24 hours. Finally, cells were stained with calcein AM (AM, live cells) and propidium iodide (PI, dead cells).

5.1.4 cell active oxygen assay

To detect ROS, cells were seeded in 96-well plates and incubated for 24 hours. Then the cells were combined with PCN (100. Mu.g.mL) ^-1 ) H at pH 6.5 ₂ O ₂ (100. Mu.M) incubated for 4 hours. Cells were then washed with PBS and incubated with DCFH-DA (20 μm) in serum-free medium for 30 min. PCN+NIR group with 808nm laser (1.0W cm) ^-2 5 min) of irradiation, and capturing a fluorescence signal of the intracellular DCF by a fluorescence microscope.

5.1.6 in vitro Blood Brain Barrier (BBB) permeation efficiency measurements

An in-vitro blood brain barrier model is established by a cross-hole culture method for the first time. 3 cells were cultured in the upper chamber to form a tight junction. At transepithelial resistance (TEER, measured using a millibattery ERS voltmeter) of greater than 200Ω cm ² PCN or PN was then added to the upper chamber and heated with an 808nm NIR laser (1.0W cm ^-2 ) With or without 5 minutes to assess BBB permeability. The samples were then incubated for 6 hours. The solution in the lower chamber was collected and analyzed using ICP-MS.

5.2 experimental results

Cell lines are commonly used to assess the ability of PCNs to cross the Blood Brain Barrier (BBB) in vitro. BBB penetration was performed using a Transwell system at 1.0 W.cm ^-2 The appropriate samples were then irradiated with 808nm laser light (FIG. 15 a). As shown in FIG. 15 b, after 6 hours of incubation, 13.40% PCN and 13.63% PN penetrated the BBB monolayer, respectively. The penetration of pcn+nir was increased by a factor of 2.18 compared to wells not irradiated with laser light. This suggests that PCN and PN are able to cross the BBB, and near infrared radiation significantly enhances this permeability. After studying the BBB penetration efficiency of PCN, cellular uptake was further studied. First, cell uptake of Cy 3-labeled PCN (Cy 3-PCN) by U87 cells at different times was examined. Flow cytometry analysis found that endocytosis of Cy3-PCN occurred in a time-dependent manner, as determined by the increase in Cy3 fluorescence intensity, cell uptake peaked after 6 hours of co-culture (c of fig. 15).

Receiving O ₂ Elicitation of efficiency PCN was studied in vitro for its ability to alleviate tumor hypoxia. Using [ Ru (dpp) 3]Cl ₂ (RDPP) as an indicator (Red fluorescence is O ₂ Quench) to detect intracellular O ₂ Horizontal. Addition of H to the culture ₂ O ₂ (100. Mu.M) to better simulate TME. Discovery and other groups (control, H ₂ O ₂ Separate group, PN separate group, PCN separate group), via PCN+H ₂ O ₂ The treated U87 cells showed the lowest fluorescence in the hypoxic environment (d of fig. 15), and the experiment was independently repeated three times. Scale bar 20m. Compared with the control group, PCN+H ₂ O ₂ The fluorescence intensity of the group was reduced 5.5-fold, indicating that PCN was at H ₂ O ₂ O generation ₂ Is shown (fig. 16).

By virtue of high cellular uptake and stable enzyme-like activity, PCN is expected to be useful in synergistic photothermal cancer treatment. As shown in fig. 15 e, after 6 hours of co-precipitation, the red fluorescence of Cy3 PCN was found to overlap with the green fluorescence of Lyso Tracker, and nuclei and lysosomes were stained with DAPI (blue) and lysosome tracer (green), respectively, which provided an acidic environment for PCN. This point was also confirmed with an electron microscope (f of fig. 15). Next, cytotoxicity and production of CDT and PTT by PCN were evaluatedObject safety. The standard CCK-8 assay showed that the cytotoxicity of PCN on HUVEC was negligible (FIG. 20 a) even at high concentrations (200. Mu.g/mL) and at 100X 10 introduction ^-6 M H ₂ O ₂ To show dose-dependent cytotoxicity (due to abnormal pathophysiological processes in the tumor microenvironment) after mimicking the tumor environment. This observation is attributable to H ₂ O ₂ ROS are produced by the Fenton-like reaction in cancer cells at relatively high levels. When further subjected to NIR laser (1.0W cm ^-2 5 minutes) the cell culture temperature was raised to 58 ℃, the viability of U87 cells was significantly reduced, and the cell killing rate was increased compared to PCN groups without NIR laser. By comparing the results of the different groups, the cytotoxic effect of the pcn+nir group was found to be best (i of fig. 15). In addition, a similar trend was also determined by combined live/dead staining (live cells, calcein AM, AM; dead cells, propidium iodide, PI) (h of FIG. 15). To confirm the correlation between toxic effects and active oxygen, a 2, 7-dichlorofluorescein diacetate (DCFH-DA) probe assay was performed to determine the production of intracellular active oxygen and to verify the mechanism of action of PCN on CDT and PTT. As shown in fig. 15 g (intracellular ROS stained by DCFH-DA (green)) and fig. 17, in PCN group, treated cells showed stronger green fluorescent signal compared to control group, and the intensity was further enhanced after NIR laser. All of these indicate that reactive oxygen species burst is initiated by addition of PCN and NIR.

6 Effect of proliferating cell Nuclear antigen on glioma in vivo

6.1 in vivo experiments

6.1.1 animals: female C57BL/6J mice and BALB/C nude mice (18-22 g,6-8 weeks old) were purchased from university of Nantong laboratory animal center. All animals were maintained in a temperature controlled environment with a 12 hour light/12 hour dark cycle, free to gain food and water. All animal experiments were performed according to the guidelines of the animal care and use institution. The study protocol was approved by the institutional animal care and use committee at university of south through.

6.1.2 in vivo PA imaging and photothermal evaluation

U87 colloid was measured using a 3.0T MRI scanner (CG MUC48-H300-AG 3.0T, china)Tumor nude mice were subjected to whole brain T2-weighted MRI (T2W-MRI). After one day, nude mice were intravenously injected with PCN (15 mg. Kg) dispersed in PBS solution through the tail curtain ^-1 ). The 3D Photoacoustic (PA) images of the entire brain were then monitored at different time points using the Vevo LAZER system (fuji film company, visual system, canada). Different concentrations (0, 125, 250, 500, 1000. Mu.g.mL) were performed in PA-free tubes prior to PA imaging ^-1 ) PA absorption and PA imaging of PCN for calibration. For in vivo photothermal evaluation, thermocouple probes were inserted into intracranial gliomas after exposing the skull region corresponding to the glioma region. 808nm laser near infrared stimulator (1.0W cm) ^-2 ) The scalp was irradiated and intracranial temperature was recorded.

6.2 experimental results

Photo Acoustic (PA) imaging was used to monitor the uptake of PCN by intracranial tumors. As shown by PA in fig. 18 a, b, intravenous PCN had time-dependent targeted accumulation in BALB/c mice carrying U87. 8 hours after intravenous injection, a clear PA signal appeared at the tumor site. The location of PA imaging is consistent with the tumor region we have pre-located by MRI. After 24 hours, the PA signal gradually decreased as PCN cleared from the tumor. These results highlight that PCN can not only achieve tumor uptake by increasing permeability and detection (EPR) effects, but also has a high potential to track glioma visualization by PA imaging capability. We studied near infrared induced PCN hyperthermia in vivo, recognizing the 8 hour tumor high accumulation. The circular skull was removed during tumor inoculation to increase the penetration of the NIR. We studied 808nm laser induced PCN in vivo hyperthermia because it has high tumor accumulation at 8 hours. The circular skull was removed during tumor inoculation to increase the penetration of the NIR (d of figure 18). After exposing the skull region corresponding to the glioma region, a thermocouple probe was inserted into the intracranial glioma (e of fig. 18). Tumor-bearing mice after intravenous PCN injection were exposed to different powers (0.5, 0.8, 1.0 W.cm) ^-2 5 min), the internal temperature of the tumor was recorded. In fact, 5 minutes of irradiation (1.0 W.cm ^-2 ) Resulting in an increase in temperature to about 50 ℃, whereas the temperature increase in the control group (normal brain tissue) was less pronounced. Correspondingly, it also has power dependencySex and time dependence (e of fig. 18).

The ability of PCN to alleviate tumor hypoxia in vivo was studied by Hypoxia Inducible Factor (HIF) -1 alpha immunofluorescent staining. Tumor hypoxia occurs in a variety of solid tumors, often leading to drug resistance and metastasis of cancer cells. Compared with the green fluorescence of the control group, less fluorescence is detected in the tumor treated by PCN, which indicates that the nano system in TME can effectively relieve tumor hypoxia. Notably, the pcn+nir group showed a significant decrease in hypoxia signal, indicating a further decrease in tumor hypoxia by mild photothermal effects (c of fig. 18).

To verify the tumor growth inhibition effect on U87 tumor-bearing mice, once daily intravenous (i.v) injections at a concentration of 15mg kg on days 14 and 16 after tumor cell implantation ^-1 (200. Mu.L) PCN. Tumors were treated with 808nm laser 8 hours after intravenous injection. T2-weighted magnetic resonance imaging (T2W-MRI) of the mouse brain was scanned at specific time points after tumor implantation. Meanwhile, three-dimensional reconstruction and imaging are performed on intracranial tumors by using 3D slicer software so as to obtain more accurate tumor sizes. As shown in f, j of fig. 18, tumors of the control group and PN group continued to grow over time. PCN groups only partially inhibited tumors. In contrast, mice in the pcn+nir group showed the greatest inhibitory effect. Pcn+nir treatment not only significantly prolonged survival, but also reduced weight loss compared to the other groups (g-i of figure 18). Brain tumor tissue was harvested on day 28 and then assessed for apoptosis. Combination treatment with PCN and NIR resulted in significant tumor apoptosis (k of fig. 18). Based on the above experiments, we found that free radical-based treatments have an inhibitory effect on tumors, yielding better therapeutic effects in combination with photothermal treatments.

In addition, the human skull has a certain thickness and is very hard. If one wants to generalize this treatment regime to clinic, it is not conceivable to resect the skull for photothermal treatment only. Clinically, it is also common for glioblastoma patients to not retain bone flaps after surgery. Based on this, the invention has devised that bone flap can be removed during glioma surgery, PCN material can be combined with hemostatic matrix (Surgiflo) commonly used by neurosurgeons to form Surgiflo@PCN, which is then applied to the tumor cavity surface after surgery, which is more convenient for post-treatment.

7ICD marker assay and DC maturation

CDT and mild PTT are critical for inducing ICD in dead cancer cells, which will synergistically increase intratumoral oxidative stress. To investigate whether PCN is capable of inducing ICD, the present invention determined several DAMP during ICD by incubating GL261 cells with PCN. GL261 cells were preincubated with PCN in mock TME, followed by gentle near infrared laser (0.8W cm ^-2 5 min) to maintain the temperature of the cell culture medium at about 45 ℃. As a result, PCN group and pcn+nir group were found to significantly induce Calreticulin (CRT) exposure in tumor cells within 6 hours, compared to control group and NIR group. Flow cytometry analysis showed that among these dead cells, the CRT exposure rate of the pcn+nir treated group was highest, with an efficiency of about 4.68 times that of the control group (f, g of fig. 19). The extracellular ATP release has the same trend (h of fig. 19). After 24 hours, the highest high mobility group protein B1 (HMGB 1) concentration was detected in the pcn+nir group (i of fig. 19). At slightly elevated temperatures, PCN-induced ICD is enhanced. In other words, PCN with multienzyme activity induces ICD under NIR control. The present invention suggests that PCNs with multienzyme activity may produce ROS, which will synergistically increase oxidative stress within tumors. To confirm our hypothesis, their performance in modulating intracellular oxidative stress was studied. As shown in fig. 20 c, the PCN-treated cells showed higher intensity of DCF intracellular fluorescence compared to the control group. After near infrared radiation, intracellular fluorescence is further enhanced. FIG. 20 c is a graph of the intensity of a laser beam (806 nm, 0.8w.cm) ^-2 ) In the case of (1), GL261 cells and PCN (100. Mu.g.mL) ^-1 ) Relative survival rate after incubation; these data suggest that we induce ROS production via PCN multienzyme activity and further enhance and coordinate this process via PTT.

Dendritic Cells (DCs) play a central role in activating the immune system, responsible for activating natural T cells. To simulate these processes, we studied PCN-induced immunogenicity by assessing the maturity of bone marrow derived dendritic cells (BMDCs) in a transwell system. GL261 tumor cells were seeded into the upper chamber, treated with PCN and/or NIR, and DC cells were seeded into the lower chamber. CD86 and CD80 staining further confirmed the maturation of DCs, which is a typical marker of mature DC surfaces. As shown in j, k of fig. 19, the percentage of mature DCs in the PCN group and the pcn+nir group increased significantly by about 1.62-fold and about 2.11-fold, respectively, compared to the percentage of mature DCs in the control group, and higher CD86/CD80 expression was observed in the pcn+nir group. Together, these results indicate that CDT and mild PTT-induced ICD show great potential in DC-T cell immune activation.

Influence of 8Surgiflo@PCN on glioma in vivo

The in vivo antitumor properties in the surgical excision model were next evaluated by the encouraging of excellent enzyme mimetic activity and immune activation properties of PCN. Surgiflo is commonly used by neurosurgery and has a multi-spatial structure that is fully absorbed by the brain (b of fig. 19). We found that a mixture of surgiflo and PCN (surgiflo@pcn) not only creates a variety of shapes for post-operative application to the tumor cavity (a of fig. 19), but also stably releases PCN. Flow cytometry analysis showed that the release of PCN and the cellular uptake rate had a stable time dependence in the co-culture system (c of fig. 19). PD-1 antibody (αpd-1) as a Food and Drug Administration (FDA) approved checkpoint inhibitor for inhibiting tumor immune evasion.

Glioma surgical resection model was constructed (a of fig. 21). First, stable luciferase transfected GL261 (Luci+GL261) cells were injected intracranially into mice to generate a mouse in situ glioma model. Mice with similar fluorescence intensities were screened using bioluminescence at day 14 post-implantation. Mice were randomly grouped prior to surgery. Mice were divided into six groups (6 per group): (1) control group, (2) αPD-1 group, (3) Surgiflo@PCN, (4) Surgiflo@PCN+αPD-1, (5) Surgiflo@PCN+NIR, (6) Surgiflo@PCN+NIR+αPD-1. Surgiflo@PCN (PCN dose of 0.75 mg.kg) for Surgiflo@PCN treatment groups (3) - (6) ^-1 Surgiflo is 7.5. Mu.L kg ^-1 ) Tiling was performed on the tumor resection cavity of each mouse. At 8 hours post-tiling, tumors of mice in group (5) and group (6) were exposed to 808nm laser light (0.8W cm ^-2 ) 5 minutes. Mice were given the following 14, 17 and 20 days of injectionAlpha PD-1 is injected intravenously with the dosage of 0.75 mg.kg ^-1 . The operation is as shown in fig. 21 a. Glioma-bearing mice were able to withstand the surgical procedure without nerve damage after surgery (b in fig. 21 is the procedure of surgical excision of the tumor from the brain of GL 261-bearing mice, b1, images of tumors formed in the brain of mice 14 days after implantation (purple arrow), b2, microsurgery excision of the tumor from brain, GBM cavity (black arrow), bone window (red arrow), b3, surgiflo@pcn in cavity, b4, excision of the tumor). A thermocouple probe was inserted into the resection cavity and used 6 hours later (c of fig. 21) after surgiflo@pcn. The internal temperature of the tumor was then recorded. In fact, 5 minutes of irradiation (0.8 W.cm ^-2 ) Resulting in a temperature rise to about 45 ℃. Accordingly, it also has power dependency and time dependency (d of fig. 21). With 808nm laser (0.8W cm) ^-2 5 min) NIR irradiation was performed in the mode shown in a of fig. 21 on mice treated 6 hours post-surgery to achieve mild PTT. The following day, the bioluminescence assay further excluded off-grade mice to ensure similar tumor residues. Bioluminescence was used to continuously monitor tumor growth. Catalytic immunotherapy with or without light PTT showed effective inhibition of tumor recurrence as shown by bioluminescence analysis. The tumor volume increase in the surgiflo@pcn group was significantly slower than in the control group or the αpd-1 group alone (P<0.01 It is notable that the Surgiflo@PCN+NIR+αPD-1 group has the strongest inhibitory effect on tumor regeneration (P<0.001 The group of mice did not even show significant tumors at the end of the study (e, g of fig. 21). Furthermore, surgiflo@pcn+nir+αpd-1 group significantly prolonged the life of tumor bearing mice, reducing the risk of tumor recurrence (f of fig. 21). The Surgiflo@PCN+alpha PD-1 group has no obvious difference from the control group in the aspects of prolonging the survival period and inhibiting the tumor, and the Surgiflo@PCN+alpha PD-1 group does have better curative effect. Histological observations of tumor-bearing brains were collected at 28 days post-implantation in the surgiflo@pcn+nir+αpd-1 group. H for brain section &E staining. A small residual Surgiflo was found in the tumor cavity. Some post-operative residual tumors were found around the tumor cavity. In further TUNEL staining, it was found that not only extensive apoptotic cells were detected at the residual tumor sites, but also in tumor foci (h of fig. 21).

To verify the mechanism of enhancing the anti-tumor immune response, the response of immune cells in brain tissue was further estimated, as well as the major immune cytokines of a portion of the mice tested (fig. 22, a is a schematic diagram of the experimental design). For the mice in group surgiflo@pcn+nir+αpd-1, the numbers of cd8+ Cytotoxic T Lymphocytes (CTLs) and cd4+ helper T lymphocytes were significantly greater than for the control group (b, d and fig. 23 of fig. 22), indicating an effective activation of the anti-tumor immune response. To further demonstrate that immunosuppressive TMEs can be reprogrammed in sub-low temperature and reverse hypoxic environments, regulatory T cells (Tregs, cd3+cd4+foxp3+) were studied. As shown in fig. 22 c, e, treg frequency was reduced to 4.15±0.26% under normal conditions, treg frequency was significantly lower in the surgiflo@pcn and surgiflo@pcn+αpd-1 groups than in the control group. The monitoring of the significant increase in the ratio of CTL to Treg cells, surgiflo@pcn+nir and surgiflo@pcn+nir+αpd-1 (f of fig. 22), suggests that they can reprogram immunosuppressive TMEs, inducing immune cell expression with anti-tumor activity. Furthermore, the surgiflo@pcn+nir+αpd-1 group showed significantly elevated levels of tumor necrosis factor- α (TNF- α), interferon- γ (IFN- γ) and interleukin-2 (IL-6) in brain tissue (g-i of fig. 22), which are key biomarkers of immune cell release in TME. These results indicate that oxidative stress and activation of high temperature induced ICDs can successfully reprogram TMEs, ultimately inhibiting postsurgical GBM regeneration.

9 in vivo biosafety assessment

Mice were intravenously injected with PCN at a dose of 30 mg/kg. The mice were sacrificed and blood was collected 14 days after implantation to obtain blood routine and serological chemistry data. 14 days after implantation, major organs including heart, liver, spleen, lung and kidney were removed and H was performed&E staining. The invention researches the influence of PCN on erythrocytes. The hemolysis results showed that even in PBS, the concentration was as high as 0.2 mg.multidot.mL ^-1 When hemolysis was negligible, it was shown to have good PCN biocompatibility (fig. 24). Second, biochemical and routine analysis of blood in vivo, in particular liver and kidney function markers, showed that 30mg kg was injected intravenously even within 2 weeks ^-1 Nor obvious hepatotoxicity and nephrotoxicity (A is shown in the order a-h of FIG. 25)ST, ALT, BUN, WBC, RBC, HCT, HGB, PLT). Third, the major organ tissues of 4 week-sacrificed mice were also subjected to hematoxylin and eosin (H&E) Staining (fig. 26). No detectable signs of damaged tissue were observed. All these results strongly indicate that intravenous PCN has negligible systemic toxicity, representing a safe approach to tumor treatment platforms.

Conclusion 10

Clinically, the treatment of GBM remains largely dependent on standard surgical resection. However, surgical resection does not completely remove tumor cells due to the wettability of gliomas, making the tumor recurrence rate extremely high. Therefore, it is particularly important to inhibit the growth of residual invasive glioma cells following GBM excision. The invention herein proposes a novel artificial enzyme PCN with enzymatic mimetic activity (including POD-like activity CAT and OXD-like activity) for use in catalytic immunotherapy. PCN can gradually increase oxidative stress in tumors by catalyzing the production of reactive oxygen species and induce tumor immunogenic death in conjunction with mild PTT, thereby reprogramming immunosuppressive TMEs and eliminating residual tumors. The present invention combines the hemostatic matrix system (surgiflo) commonly used by neurosurgeons with PCN (surgiflo@pcn) to enable the treatment of glioma infiltration during surgery. This design has the following advantages:

(1) The surgiflo@PCN is applied to the tumor cavity in situ under the condition that the original characteristic of surgiflo is not changed, so that the concentration and the acting time of the PCN are greatly increased. (2) After removal of a small "bone window" (which is also in line with clinical post-operative treatment), the efficiency of mild photothermal therapy can be conveniently improved. (3) Surgiflo is a mature hemostatic material that can greatly reduce post-operative bleeding and other complications in clinical applications, particularly in microsurgery. This strategy would provide new anti-tumor immunity opportunities for patients undergoing brain tumor resection, inhibiting postoperative tumor recurrence, which might be translated into artificial enzymes for clinical use in future.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims (6)

1. The application of the composite material for inhibiting postoperative tumor recurrence in preparing a medical material for inhibiting postoperative tumor recurrence is characterized in that the preparation method of the composite material comprises the following steps:

Dissolving 10-15mg of polyvinylpyrrolidone in 1-2mL of deionized water to obtain PVP solution, adding a mixed solution of copper chloride and palladium (II) potassium chloride into the PVP solution, stirring at 25-28 ℃ for 1-2min, adding 1-1.5mL of ascorbic acid, performing ultrasonic treatment for 15-20min, and dialyzing with deionized water through a dialysis bag with a molecular weight cut-off of 10-15kDa for 2-3 days to obtain the composite material for inhibiting postoperative tumor recurrence;

the mixed solution is a mixed solution of 0.3-0.5mL of 20mM copper chloride and 0.7-1mL of 20mM palladium (II) chloride potassium;

the particle size of the composite material is 30-40nm;

the tumor includes glioma.

2. The use according to claim 1, wherein the composite material acts to inhibit postoperative tumor recurrence in combination with gelatin Surgiflo, which is an absorbable hemostatic fluid.

3. The use according to claim 2, wherein the composite material is combined with gelatin Surgiflo which can absorb hemostatic fluid and is injected into a patient, and the combination of chemo-and photothermal therapy is used for generating active oxygen, enhancing oxidative stress of tumor cells, killing tumor cells and inhibiting postoperative tumor recurrence.

4. The use according to claim 2, wherein the composite material is injected into a patient in combination with gelatin Surgiflo, which absorbs hemostatic fluid, to induce immunogenic cell death, reverse immunosuppressive tumor microenvironment, and enhance anti-tumor immune response to inhibit postoperative tumor recurrence.

5. A medical material for inhibiting postoperative tumor recurrence, comprising the composite material of claim 2 in combination with a material that absorbs hemostatic fluid gelatin Surgiflo.

6. The medical material of claim 5, wherein the tumor comprises a glioma.