CN115531542A

CN115531542A - Composite material for inhibiting postoperative tumor recurrence and preparation method and application thereof

Info

Publication number: CN115531542A
Application number: CN202211213229.4A
Authority: CN
Inventors: 施炜; 聂德康; 刘倩倩; 黄容琴; 王�义
Original assignee: Affiliated Hospital of Nantong University
Current assignee: Affiliated Hospital of Nantong University
Priority date: 2022-09-29
Filing date: 2022-09-29
Publication date: 2022-12-30
Anticipated expiration: 2042-09-29
Also published as: NL2033379B1; CN115531542B

Abstract

The invention discloses a composite material for inhibiting postoperative tumor recurrence and 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 of: and adding a mixed solution of copper chloride and potassium palladium (II) chloride into a 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 the oxidative stress in tumors, can directly kill glioma cells through CDT and PTT, can also enhance anti-glioma immunity and indirectly kill glioma, and provides an effective photo-thermal immunotherapy strategy of 'one stone two birds' for glioma patients.

Description

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

Technical Field

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

Background

Glioblastoma (GBM) is the most common primary brain tumor clinically with poor prognosis, high recurrence and mortality. The median overall survival of patients with primary glioblastoma is only around 14.6 months. GBM treatment remains largely dependent 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 therapeutic approaches for recurrent GBM is crucial for cancer patients. Immunotherapy, considered one of the most effective methods for treating cancer, has proven to have great potential in an increasing number of malignancies. With intensive research into the Central Nervous System (CNS), researchers have found that microglia play a dominant 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 much fewer T lymphocytes. Macrophages play a very important role in the development, progression, invasion and immune escape of tumors. Therefore, it is important to alter the TME, relieve the immunosuppressed state, and increase the entry of activated T lymphocytes into the TME.

Immunogenic Cell Death (ICD) is a cell death pathway that has recently become the focus of therapy-induced anti-tumor immunity. As ICDs are received, the dead tumor cells release damage-associated molecular patterns (DAMPs) and tumor-associated antigens (TAAs). Major 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). In the initial phase of ICD induction, CRT translocates to the cell surface, heat shock proteins act as "eat me" signals, and in the middle of cell death, extracellular released or secreted ATP acts as an effective "find me" signal. Similarly, in the late stages of cell death, extracellular release of HMGB1 serves as an immunostimulatory "danger" signal. First, these ICD-derived DAMPs recruit and activate the innate immune system by stimulating antigen presentation by Dendritic Cells (DCs), and then achieve processing and presentation to the T cell receptor. Second, these activated T cells subsequently infiltrate the tumor in large numbers and help eliminate residual cancer cells. At the same time, TAAs released or exposed from dead cancer cells may further enhance antigen-specific T cell responses.

With the development of biomedical technology, various therapeutic modalities for ICD inducers have been disclosed, including but not limited to: conventional chemotherapy (anthracyclines, oxaliplatin, etc.), radiation therapy and photodynamic therapy. This provides a means and challenge for treatment of gliomas. The standard treatment for gliomas is surgical resection followed by radiotherapy and chemotherapy with TMZ (a second generation oral alkylating agent, but not ICD induction alone). For GBM, the treatment effect is poor, the side effect is large, and most GBM still needs secondary operation. It is well known that tumors have unique microenvironments, such as mild acidity, high hydrogen peroxide (50-100X 10) ^-6 M) and hypoxia. Hypoxia can induce tumor cell metastasis, ultimately resulting in decreased efficacy. To address this problem, H has been explored for tumor therapy ₂ O ₂ A responsive nanoenzyme. The nanoenzyme has Peroxidase (POD) and Oxidase (OXD) like activities, and can convert H under TME ₂ O ₂ And O ₂ Decomposed into Reactive Oxygen Species (ROS). In addition, photothermal effects may enhance both POD and OXD-like activity, thereby generating more ROS. This is a perfect combination of CDT and PTT. Notably, oxidative stress local CDT/PTT may meet ROS-based oxidative stress criteria. On the one hand, powerful ROS and PTT can kill tumor cells. On the other hand, oxygen based on ROSThe chemical stress may further induce the ICD to produce secondary damage to tumor cells. These findings open new avenues for designing artificial enzymes with ICD-related immunotherapy modalities.

The nanometer enzyme is an important component of nanometer materials and has the characteristics of multiple functions, high stability, low cost and the like. These advantages make it superior to traditional therapies in practical applications, compared to natural enzymes. However, the nano-enzyme is high in cost, and the transition metal with low cost is usually selected to manufacture the bimetal composite material, so that 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 and 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 tumor killing, 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 purpose, the invention provides the following scheme:

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

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

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

The invention also provides a composite material for inhibiting postoperative tumor recurrence, which is obtained according to the preparation method.

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

Further, the composite material in combination with the absorbable hemostatic fluid gelatin Surgiflo exerts efficacy in inhibiting postoperative tumor recurrence.

Further, the composite material is combined with absorbable hemostatic fluid gelatin Surgiflo and injected into a patient, and activated oxygen is generated by means of chemodynamic therapy and photothermal therapy, so that oxidative stress of tumor cells is enhanced, the tumor cells are killed, and postoperative tumor recurrence is inhibited.

Further, the composite material is combined with absorbable hemostatic fluid gelatin Surgiflo and injected into a patient, immunogenic cell death is induced, an immunosuppressive tumor microenvironment is reversed, and anti-tumor immune response is enhanced, so that postoperative tumor recurrence is inhibited.

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

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, PCN catalyzed ROS enhance oxidative stress in tumor and further induce ICD in photothermal therapy (PTT), the composite material is combined with absorbable hemostatic fluid gelatin Surgiflo becomes hemostatic matrix system (Surgiflo @ PCN), surgiflo @ PCN directly kills glioma cells through chemokinetic therapy (CDT) and PTT, then ICD is induced, immunosuppressive Tumor Microenvironment (TME) is reversed, and anti-tumor immune response is enhanced, so that residual glioma cells are eliminated and recurrence is prevented.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.

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

FIG. 2 is a TEM image of a PN;

FIG. 3 is STEM and EDS mapping images 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 photograph of the stable dispersion of PCN and PN in water;

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

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

FIG. 8 is a characterization of OXD-like activity of PCN; a is TMB in N ₂ Air and oxygen are directly oxidized by PCN; b is the OXD-like activity change of PCN and PN at different temperatures; c is the absorption spectrum and visible color change of TMB after 10min incubation in the presence of different concentrations of PCN, where 1: 0. Mu.g/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. Mu.g/mL ^-1 1-8 all contain TMB, M does not contain TMB; d is the OXD-like activity change of PCN and PN at different pH;

FIG. 9 shows the enzymatic kinetics of PCNThe chemical result is that a is the result of measuring the OXD-like activity of PCN and PN catalyzed and oxidized by TMB; b is PCN and PN at H ₂ O ₂ The catalytic activity of (1); c is comparison of catalase-like activities of PCN and PN; d is the temperature change of PCN solutions with different concentrations under the irradiation of laser with 808 nm; e is (1W cm) under 808nm laser irradiation only ^-2 ) PCN versus deionized water heating curve; f is the photo-thermal stability research result of the PCN under 808nm laser irradiation in the heating-cooling process of 5 cycles; g is a schematic diagram of the activity of a PCN-like enzyme; h is an image of PCN solution with different concentrations; i is the linear relationship between signal intensity and PCN concentration;

FIG. 10 shows POD-like activity characteristics of PCNs; 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 is a CAT-like activity profile of PCN; a is H ₂ O ₂ And PCN at different concentrations to generate a photograph of oxygen, where 1: 0. Mu.g/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 containing 100mM H ₂ O ₂ The red color of the bottle is marked as an oxygen bubble; b is PCN and PN pair H at different times ₂ O ₂ The influence of oxygen generated by decomposition; c is the catalytic activity of PCN and PN under different pH values;

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

FIG. 13 shows the three enzyme-like activity assays for OXD, POD and CAT of PCN; a is PCN and FeSO ₄ And CuSO ₄ Comparative photograph of color change of OXD-like Activity of (b) is PCN, feSO ₄ And CuSO ₄ Comparative statistical plots of OXD-like activity of (a); c is PCN, feSO ₄ And CuSO ₄ POD-like activity ofA color change contrast photograph of nature; d is PCN, feSO ₄ And CuSO ₄ A POD-like activity comparison histogram of (a); e is PCN, feCl ₃ And CuSO ₄ Comparative photographs of color change of CAT-like activity of (1); f is PCN, feCl ₃ And CuSO ₄ CAT-like activity comparison statistical chart of (A);

FIG. 14 shows photothermal properties of PCN; a is laser irradiation (1.0W. Cm) at 808nm ^-2 ) Next, the temperature of the PCN and PN solutions of the same concentration was varied; b is measured at 808nm under laser irradiation with different power densities (1.0, 0.8, 0.5 w-cm) ^-2 ) Temperature change of the PCN solution of (a);

FIG. 15 is the ability of PCN to cross the Blood Brain Barrier (BBB) in vitro; a is a schematic diagram of a BBB permeation model using a Transwell system; b is PN, PCN and PCN + NIR (808nm, 0.1W cm) ^-1 ) BBB permeability comparison of (a); c is the result of quantitative analysis of PCN uptake by U87 cells by flow cytometry; d is O in U87 cells under the hypoxic environment ₂ The generated fluorescence image; e is the location of PCN in U87 cells; e is a TEM image of PCN in U87 cells after 6 hours incubation; g is the intracellular ROS level in U87 cells after various treatments; 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 of U87 cells after different treatments; group 1 control, group 2 NIR only, group 3 PN, group 4 PCN and group 5 PCN + NIR; 100 g/mL of PCN or PN ^-1 Near infrared laser of 808nm,1w cm ^-2 ，5min；

FIG. 16 shows the intracellular production of O in different groups ₂ Comparing the fluorescence intensities of (a);

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

FIG. 18 is a graph of Photoacoustic (PA) imaging monitoring of PCN uptake by intracranial tumors; a is in vivo PA imaging of U87 tumor-bearing mice after intravenous injection of PCN; b is the time-dependent tumor PA signal based on the PA imaging data in a; c fluorescence images detected in the tumors treated in each group, nuclei and hypoxic regions are shown with DAPI (blue) and anti-HIF-1. Alpha. Antibody (green); d is the small tumor load of U87 after the surgical skull resection and tumor inoculationPerformed on mice, black circles indicate surgical bone windows; e is laser irradiation (1.0W cm) of U87 tumor at 808nm ^-2 Temperature change curve at 5 min); f is T2W-MRI imaging of U87 tumor-bearing mice after different treatments (including control group, PN, PCN and PCN + NIR); yellow circles indicate tumors; g is the average tumor growth curve of 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 the size of the intracranial tumor; k is TUNEL staining of glioma cell apoptosis from different treatment groups;

FIG. 19 a is representative photographs of PCN, surgiflo, and Surgiflo @ PCN of various geometries; b is a representative scanning electron microscope image of Surgiflo and Surgiflo @ pcn; the scale bar is 100 mu m; c-d is the cellular uptake of Surgiflo @ Cy3-PCN by GL261 cells quantified by flow cytometry using the Transwell system; e is STEM image and EDS mapping image of Surgiflo @ PCN, scale: 10 μm; f is the in vitro CRT exposure of GL261 cells after different treatments as determined by flow cytometry in mock TME; g is the quantitative percentage of CRT exposure in different groups; h is extracellular ATP secretion of GL261 cells assessed using ATP kit; i is the HMGB1 drug release profile detected using the enzyme linked immunosorbent assay (elisaxit); j is flow cytometric analysis of DC maturation after 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 survival of normal cells (HUVECs) with different concentrations of PCN; b is the presence or absence of laser irradiation (808nm, 0.8w.cm) in a simulated TME ^-2 ) In the case of (1), GL261 cells and PCN (100. Mu.g. ML) ^-1 ) Relative survival after incubation; c is the intracellular ROS level in GL261 cells treated in each group, PCN or PN:100 g.mL ^-1 Near infrared laser of 808nm, 0.8w.cm ^-2 ，5min；

FIG. 21 is the results of a Surgiflo @ PCN test for suppressing postoperative recurrence of glioblastoma; a is a schematic diagram of experimental design; b is the process of surgical resection of a tumor from the brain of a mouse carrying GL261, b1, images of tumors (purple arrows) formed in the mouse brain 14 days after implantation; b2, microsurgical resection of tumors from brain, GBM cavity (black arrow), bone window (red arrow); b3, surgiflo @ PCN in the cavity; b4, removing the tumor; c-d is the operation of irradiating the resection cavity with laser at 808nm by using different laser power densities and the temperature change curve of the tumor; e, g is a representative bioluminescence analysis image (e) and quantified signal intensity (g) (n =3 biologically independent animals per group); f is survival curve of differently treated GL261 bearing mice (n =6 mice per group); h are representative HE and Tunel staining images of brain tissue from mice bearing GL261 showing tumor resection cavities, blue circles indicate tumor cavities; yellow circles indicate infiltrated residual tumors; red circles indicate residual Surgiflo;

FIG. 22 is an in vivo mechanism of enhanced antitumor immune response by brain tissue; a is a schematic diagram of experimental design; b, d are representative flow cytometry analyses of T cell infiltration on CD3+ cells in brain tissue with glioma after resection (b) and quantification results in different groups (d); c, e are representative flow cytometric 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 level of secretion of IL-6, TNF- α and IFN- γ in brain tissue after each treatment;

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

FIG. 24 is a graph showing the hemolysis rate of PCN nanoenzyme at different concentrations;

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

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

Detailed Description

Preparation of 1 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 potassium palladium (II) chloride (K) ₂ PdCl ₄ ) (0.7 mL) of the mixed solution at room temperature (25 ℃ C.)) Stirring for 1min, quickly adding 1mL of ascorbic acid into the reaction system, finally performing ultrasonic treatment for 15min, dialyzing for 3 days by using deionized water, wherein the cut-off molecular weight of a dialysis bag is 10KDa, and obtaining the product, namely the PCN composite material.

Pure Palladium Nanoparticles (PN) were obtained by the above method using deionized water instead of copper chloride.

Material characteristics of 2 PCN

2.1 Experimental methods

Transmission Electron Microscopy (TEM), high Resolution TEM (HRTEM), scanning Transmission Electron Microscopy (STEM), and energy dispersive X-ray (EDX) mapping of the catalyst were performed on a JEM-2100 electron microscope (JEOL, japan) with an acceleration voltage of 200 kV. The field emission Scanning Electron Microscope (SEM) observation was performed on a Regulus8230 microscope (hitachi, japan). X-ray diffraction (XRD) analysis was obtained on a D/MAX 2550VB/PC diffractometer (university of Japan). X-ray photoelectron spectroscopy (XPS) was performed on the RBD upgraded PHIE5000C ESCA system (Perkinelle, USA). Fourier transform infrared spectroscopy (FTIR) in the range of 400-4000cm-1 was recorded on a VERTEX70 spectrometer (Bruker, germany) spectrometer. Thermogravimetric analysis (TGA) was performed using a discover TGA550 system (TA Instruments, USA) at a temperature range of 30-900 ℃ under an air atmosphere. Surface area was calculated from the adsorption data using Langmuir and Brunauer-Emmett-Teller (BET) methods. The raman spectra were captured on a Renishaw (Renishaw, uk) confocal microscope raman spectrometer.

2.2 results of the experiment

PCN is Cu assisted by ultrasound in the presence of polyvinylpyrrolidone (PVP) and ascorbic acid ²⁺ And Pd ²⁺ Is easily synthesized, which is a small-grained nanoparticle assembly of 30nm to 40nm, as shown in fig. 1, a, which is a Transmission Electron Microscope (TEM) image and an energy dispersive X-ray spectroscopy (EDS) image of PCN, mesopores are observed, and pores are not observed in PN (fig. 2). At the same time, the apparent lattice fringes in the High Resolution Transmission Electron Microscope (HRTEM) images verify that the crystalline nanoparticles, in particular the lattice distance of 0.22nm, imply the typical (111) crystallographic plane of the PCN alloy (fig. 1, panel b). Accordingly, scanning Transmission Electron Microscope (STEM) images and EDS mapsThe homogeneous distribution of copper elements in the Pd matrix shown by the gamma ray image confirms the PCN alloy, which assembles well into homogeneous porous nanoparticles (fig. 1, panel c). This alloying effect can also be revealed by X-ray diffraction (XRD) patterns and X-ray photoelectron spectroscopy (XPS) spectra, where the much broader diffraction of PCN than PN implies small grains with abundant defects, and the Pd 3d binding energy of PCN is shifted negatively compared to PN indicating d-band center down-regulation by Cu-doped Pd (see d-e in fig. 1), and furthermore, additional Cu XPS signals, i.e., cu2p 3/2 and Cu2p 1/2, appear in PCN other than PN, which is in good agreement with the EDS mapping results, consolidating the alloy formed by Pd and Cu. Thus, the porous and defect structure with down-regulated d-band center facilitates the catalytic application of PCN because of their 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 profiles (fig. 3), where 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, whose Dynamic Light Scattering (DLS) particle size is only slightly larger than the TEM-measured particle size due to polymer-mediated hydrophilization (a-b of fig. 4). Thereafter, the synthesized nanoparticles can be well dispersed in various stimulated physiological media without precipitation in water even after centrifugation 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 can be well dispersed in various solvents, such as Simulated Body Fluid (SBF), PBS, and deionized water, indicating that PCN has good stability in these media (fig. 6). Importantly, it decomposed very little even at high hydrogen peroxide concentrations (fig. 7).

OXD, POD and CAT-like Activity and kinetic assays for 3PCN

3.1 Experimental methods

3.1.1 detection of OXD-like Activity

Oxidation of 3,3', 5' -Tetramethylbenzidine (TMB) in NaAc buffer (0.1M, pH 5.0) by PCN produced a blue signal. Kinetic measurements of the PCN oxidase reaction were evaluated at 652nm by a multifunctional microplate reader (Thermo Scientific, varioskan Lux, usa). Typically, 10. Mu.L of PCN (final concentration 7.5. Mu.g)·mL ^-1 ) Add 200. Mu.L NaAc buffer containing 10. Mu.L TMB (final concentration 0.416 mM) to show color reaction. Steady State kinetic analysis was performed 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. ML) ^-1 In DMSO), kinetic analysis of PCN was performed with TMB as substrate. The absorbance of all reactions was monitored at different reaction times and the michaelis constants were calculated from michaelis-menten saturation curves 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, 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 POD-like activity assays of PCN were performed by a multifunctional microplate reader (Thermo Scientific, varioskan Lux, USA) at 652nm in the presence of PCN. In a typical experiment, 10. Mu.L of PCN (final concentration 7.5. Mu.g.mL) ^-1 ) Added to 200. Mu.L of NaAc buffer containing 10. Mu.L of TMB (final concentration 0.416 mM) and 10. Mu.L of H ₂ O ₂ (final concentration 50 mM) to show a color 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 in TMB. By adding 10. Mu.L of TMB (2 mg. ML) ^-1 In DMSO) and varying amounts (1, 2, 4, 6, 8, 10, 12, 14, 16, 20, 30, 40 μ L) of H ₂ O ₂ Solution (1M) in H ₂ O ₂ Kinetic measurements of PCN were performed for the substrates. By adding 10. Mu.L of 1M H ₂ O ₂ And various amounts (1, 2, 4, 6, 8, 10, 12.5, 15, 17.5, 20. Mu.L) of TMB solution (2 mg. ML) ^-1 DMSO) was performed for the PCN kinetic assay using TMB as a substrate. All reactions were monitored by measuring absorbance at different reaction times and the michaelis constants were calculated from michaelis-menten saturation curves by GraphPad Prism 7.0 (GraphPad software). POD-like Activity of PCN detected at various temperatures of 25 ℃ to 55 ℃Temperature dependence, the pH dependence was detected in different buffer solutions with pH values between 2 and 9. For comparison, the POD activity of PN was also measured.

3.1.3CAT-like Activity

O measurement by using a specific oxygen electrode on a multiparameter analyzer (JPSJ-606L, leici China) ₂ And generating and measuring CAT-like activity of the PCN. In general, 25. Mu.L of PCN (final concentration: 7.5. Mu.g. ML) ^-1 ) Adding a solution containing 100. Mu.L of 0.1M H ₂ O ₂ 1mL of PBS buffer (0.1M pH 7.0). Measuring O at different time points ₂ And (4) generating. 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. Mie-manten constants were calculated from the mie-manten saturation curves by GraphPad Prism 7.0 (GraphPad software). The pH dependence was examined in different buffer solutions with pH values of 3 to 9. For comparison, the CAT-like activity of PN was also measured.

To detect H ₂ O ₂ Consumption in direction H ₂ O ₂ To the (10 mM) solution was added 10. Mu.L of PCN (final concentration 7.5. Mu.g. ML) ^-1 ). After five minutes, the solution was centrifuged and the remaining H was recorded by UV-vis spectrophotometer (Shimadzu UV-2600) at 240nm ₂ O ₂ The absorbance of (2). Evaluation of H against calibration Curve ₂ O ₂ By recording various known concentrations of H at 240nm ₂ O ₂ (1-50 mM) absorbance.

3.2 results of the experiment

Nanolase inducible O with OXD-like Activity or POD-like Activity ₂ Or H ₂ O ₂ Active oxygen is generated by decomposition, thereby leading to the catalytic oxidation of 3,3', 5' -Tetramethylbenzidine (TMB) to form a blue product (oxTMB) with the absorption of 652 nm. The present inventors studied 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, under the same conditions, PN is less catalytically active. TMB catalytic oxidation has also been foundThe Vmax value of PCN increased 1.6-fold relative to PN (a of fig. 9), and these results confirm that PCN has higher catalytic activity than PN. Also evaluated pure O ₂ Air and N ₂ OXD-like activity in (1). It was found that pure O is compared with air ₂ Is significantly increased, N ₂ The enzyme-catalyzed reactions in the atmosphere were significantly reduced (a of fig. 8). The role of oxygen in OXD-like activity was further confirmed. Fig. 8 c is an absorbance spectrum and visible color change of TMB (0.416 mM) after 10 minutes incubation in the presence of different concentrations of PCN (0.1 m naac buffer, pH 5.0), where 1: 0. Mu.g/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. Mu.g/mL ^-1 1-8 all contain TMB, M does not contain TMB; data are shown as mean ± standard deviation (n =3 biologically independent experiments).

TMB in H ₂ O ₂ POD-like activity of PCN and PN was studied in the presence of H ₂ O ₂ Decomposition generates ROS (e.g., OH). Due to the presence of abundant active catalytic sites, PCN showed higher POD-like activity compared to PN (a of fig. 10). The catalytic activity is also pH and temperature dependent (c, d of fig. 10). The maximum pH and temperature for PCN and PN catalytic activity were pH 5.0 and 40 ℃, respectively. With H ₂ O ₂ And TMB as a substrate, a typical Michaelis equation of kinetics is established. PCN and PN showed significant catalytic activity in both substrates (b of fig. 9 and b of fig. 10). In contrast to PN, TMB and H ₂ O ₂ The Vmax values of the upper PCN increased 4.6-fold and 6.1-fold, respectively. Clearly, PCN has a high catalase-like activity. Thus, all of these results further demonstrate that PCN can catalyze H as an effective enzyme-like activity ₂ O ₂ And generates reactive oxygen species toxic to cancer cells.

To study H ₂ O ₂ Decomposition to O ₂ And water, and measuring O in the solution by a dissolved oxygen meter ₂ And (4) concentration. We have found H ₂ O ₂ O of solution ₂ Concentration dependent on PCN concentrationIncreases rapidly, with the optimum pH in the neutral range (pH 7.0) (fig. 11 a, c). Further investigation of PN revealed that PN was less active than PCN (FIG. 11 b). PCN decomposes H as shown in c of FIG. 9 ₂ O ₂ Mie kinetics were followed. In the comparison between PCN and PN, the Vmax value of PCN is slightly higher than PN, however, its Km value is much lower than PN, which means for H ₂ O ₂ High affinity and high catalase-like activity. Furthermore, by measuring H at 240nm ₂ O ₂ Monitoring H in the PCN-catalyzed reaction ₂ O ₂ The concentration was varied with time and a standard curve was established (a, b of FIG. 12). We observed H ₂ O ₂ The concentration continued to decrease within 5 minutes after the reaction, which was accompanied by 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 the classical Fenton reaction ions, it is found that PCN has higher catalytic activity under the same molar concentration environment, which not only embodies the advantages of the nano material, but also verifies that PCN has higher catalytic performance (a-f of FIG. 13). The results show that the reactivity of PCN is superior to that of PN, demonstrating that bimetallic PCN has higher catalytic activity than monometallic PN. PCN can act as a peroxisome mimetic, and thus can modulate cellular ROS.

Evaluation of photothermal Effect of 4 PCN

4.1 evaluation method

First, PCN (0, 12.5, 25, 50, 100. Mu.g.mL) was prepared at various concentrations ^-1 ) Irradiating with 808nm laser (200 μ L) in a glass bottle (Vicko, china, ltd.) ^-2 For 10 minutes. During irradiation, the temperature change of the solution was monitored by a thermocouple (provided by the intelligent drug delivery focus laboratory at the university of fondand). Next, the concentration was adjusted to 100. Mu.g.mL ^-1 The PCN solution of (a) was used for further photothermal property characterization. Using 808nm laser at different power densities (0.5W cm) ^-2 、0.8W·cm ^-3 And 1.0W·cm ^-4 ) The PCN was irradiated for 10min. Temperature changes were monitored during the irradiation process. Under 808nm laser irradiation, a PCN solution (100. Mu.g.mL) ^-1 1 mL) were performed for five cycles of real-time temperature measurement. Each cycle included 5 minutes of irradiation followed by a5 minute cool down period.

4.2 results of the experiment

Significant concentration-dependent and laser power-dependent photothermal effects of PCN were observed within 5 minutes (d of fig. 9 and b of fig. 14), and infrared thermography showed that at 100 μ g · mL ^-1 The temperature rose from 24.2 c to 58.4 c, while no significant temperature change was observed in the deionized water (e of fig. 9). Then, the photothermal 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 a PCN-like enzyme. In addition, the same concentration (100. Mu.g.mL) was investigated ^-1 ) The photo-thermal property (delta is approximately equal to 27.1 ℃) of PN is slightly higher than that (delta is approximately equal to 24.2 ℃) of PN (1.0W cm ^-2 5 minutes) (fig. 14 a). Furthermore, PTT binding to reactive oxygen species production has been demonstrated to be an effective strategy for killing cancer cells. In addition, PCN can produce a strong photoacoustic signal. As shown in h and i of FIG. 9, the PCN in aqueous solution is linearly related to the PA signal (R) ² = 0.978), data shown as mean ± standard deviation (n =3 biologically independent experiments), concentration increase ranged from 0 to 1000 μ g · mL ^-1 . Therefore, PCN can be used as a PA contrast agent to guide the location and timing of irradiation, and PCN contrast PA imaging offers great potential for tumor visualization.

5 Effect of proliferating cell nuclear antigens on in vitro tumor cells

5.1 design of the experiment

5.1.1 cell culture: u87, GL261 and Luci + GL261 cells were purchased from Xin-Tech, inc., shanghai. The cell lines were cultured in complete Dulbecco's Modified Eagle's Medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 1% penicillin G sodium, and 1% streptomycin sulfate. 95% air and 5% CO of cells at 37 ℃ ₂ Incubations were performed in a humidified atmosphere and subcultured by addition of 0.25% trypsin and complete DMEM.

5.1.2 cellular uptake and subcellular distribution

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

The U87 cells were digested to obtain a cell suspension, which was seeded into each well 10 ⁵ In 6-well plates of individual cells. After 24 hours of culture, the medium was replaced, and the volume of the medium was 100. Mu.g/mL per well ^-1 Cy3-PCN was added at the concentration of (3). After 0, 1, 2, 4, 6 hours, the cells were washed twice and then analyzed by flow cytometry (BD FACS Calibur, usa).

To follow the subcellular distribution of PCN, U87 cells were cultured with Cy 3PCN for 6h. To visualize lysosomes, cells were stained with Lyso Tracker Green (Beyotime Biotechnology, china) and Hoechst for 30 min at RT, respectively. Cells were washed with PBS before observation under a fluorescent microscope (zeiss, germany).

5.1.3 in vitro cytotoxicity assay

Biocompatibility testing of PCN was performed on HUVEC. Cells were plated in 96-well plates at 0.8X 10 per well ⁴ Density culture of individual cells and 5% CO at 37 ℃% ₂ 100 μ L of DMEM medium for 24 hours. The continuous concentration (0, 25, 50, 100, 200. Mu.g.mL) ^-1 ) The PCN of (2) is added to the above medium. After 24 hours of incubation, 10. Mu.L of CCK-8 was added to each well. Plates were incubated at 37 ℃ for 2 hours and then measured in a multimode microplate reader (BioTek, synergy2, USA) at 450nm optical density.

For cytotoxicity assays, U87 cell suspensions were administered at 0.8 × 10 per well ⁴ The density of individual cells (in 100. Mu.L DMEM medium) was seeded in 96-well plates. Subsequently, PCN (0, 12.5, 25, 50, 100. Mu.g.mL) ^-1 ) And H ₂ O ₂ (100. Mu.M) Medium (pH 6.5) was added. After 6 hours, use 100. Mu.g.mL ^-1 U87 cells incubated with PCN were irradiated with 808nm laser (1.0W. Cm) ^-2 ) The irradiation was carried out for 5 minutes. Then, the cells were further incubated for 24 hours.Finally, cell viability was assessed by the CCK-8 kit assay according to the manufacturer's instructions.

For live/dead staining, U87 cells were plated at 2 × 10 per well ⁴ The density of individual cells was seeded in 24-well plates. U87 cells were compared with control, NIR (808nm, 1.0W.cm) ^-2 5 min), PN (100. Mu.g.mL) ^-1 )、PCN(100μM mL ^-1 ) And H of PCN + NIR at pH 6.5 ₂ O ₂ (100. Mu.M) treatment under co-incubation. Then, the cells were further incubated for 24 hours. Finally, cells were stained with calcein AM (AM, live cells) and propidium iodide (PI, dead cells).

5.1.4 cellular active oxygen assay

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

5.1.6 in vitro measurement of Blood Brain Barrier (BBB) permeation efficiency

The in vitro blood brain barrier model is established by adopting a cross-pore culture method for the first time. 3 cells were cultured in the upper chamber to form tight junctions. Achieves a trans-epithelial resistance (TEER, measured using a millicell ERS volt ohm meter) of greater than 200 Ω cm ² Thereafter, PCN or PN was added to the upper chamber and NIR laser (1.0W cm) at 808nm ^-2 ) With or without 5 minutes of irradiation 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 results of the experiment

Cell lines are commonly used to evaluate the ability of PCN to cross the Blood Brain Barrier (BBB) in vitro. BBB penetration Using the Transwell System at 1.0 W.cm ^-2 Next, a suitable sample was irradiated with 808nm laser light (FIG. 15, a). As shown in fig. 15 b, 13.40% PCN and 13.63% PN penetrated the BBB monolayer, respectively, after 6 hours of culture. The penetration of PCN + NIR was increased by 2.18 compared to the non-laser irradiated poresAnd (4) multiplying. This indicates that PCN and PN are able to cross the BBB and that near infrared radiation significantly enhances this permeability. After studying the BBB penetration efficiency of PCN, cellular uptake was further studied. First, the cellular uptake of Cy 3-labeled PCN (Cy 3-PCN) by U87 cells at different stages was examined. Flow cytometry analysis revealed that endocytosis of Cy3-PCN occurred in a time-dependent manner, as determined by the increase in Cy3 fluorescence intensity, with cellular uptake peaking after 6 hours of co-culture (c of fig. 15).

To receive O ₂ Inspired by the efficiency of production, PCN was studied for its ability to alleviate tumor hypoxia in vitro. Using [ Ru (dpp) 3]Cl ₂ (RDPP) as indicator (Red fluorescence by O) ₂ Quenching) to detect intracellular O ₂ And (4) horizontal. Addition of H to the culture ₂ O ₂ (100. Mu.M) to better mimic TME. Found in the other groups (control group, H) ₂ O ₂ Single group, PN single group, PCN single group) compared with 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 repeated three times independently. Scale bar: 20m. PCN + H compared to control group ₂ O ₂ The fluorescence intensity of the group decreased 5.5 times, indicating that PCN was at H ₂ O ₂ Generation of O ₂ Catalytic ability in (fig. 16).

With high cellular uptake and stable enzyme-like activity, PCN is expected to be useful in synergistic photothermal cancer therapy. As shown in fig. 15 e, 6 hours after co-precipitation, the red fluorescence of Cy 3PCN was found to overlap with the green fluorescence of Lyso Tracker, and the nucleus and lysosome were stained with DAPI (blue) and lysosomal tracer (green), respectively, which provided an acidic environment for PCN. This view was also confirmed with an electron microscope (f of fig. 15). Next, PCN was evaluated for cytotoxicity and biosafety against CDT and PTT. Standard CCK-8 assays showed that the cytotoxicity of PCN against HUVEC was negligible even at high concentrations (200. Mu.g/mL) (a in FIG. 20) and 100X 10 at the time of introduction ^-6 M H ₂ O ₂ To mimic the tumor environment and then show dose-dependent cytotoxicity (due to abnormal pathophysiological processes in the tumor microenvironment). This observation can be attributed to H ₂ O ₂ Passage in cancer cells at relatively high levelsFenton-like reactions produce ROS. When further NIR laser (1.0W cm) was performed ^-2 5 minutes), the cell culture temperature was increased to 58 ℃, the survival rate of U87 cells was significantly decreased, and the cell killing rate was increased compared to the PCN group without NIR laser. The best cytotoxic effect of the PCN + NIR group was found by comparing the results of the different groups (fig. 15 i). Furthermore, 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 reactive oxygen species, 2, 7-dichlorofluorescein diacetate (DCFH-DA) probe assays were performed to determine intracellular reactive oxygen species production and to validate the mechanism of action of PCN on CDT and PTT. As shown in g of fig. 15 (intracellular ROS stained by DCFH-DA (green)) and fig. 17, in the PCN group, the treated cells showed stronger green fluorescence signals and the intensity after NIR laser was further enhanced compared to the control group. All of these indicate that the active oxygen burst is initiated by the addition of PCN and NIR.

6 Effect of proliferating cell nuclear antigen on in vivo glioma

6.1 in vivo experiments

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

6.1.2 in vivo PA imaging and photothermal evaluation

U87 glioma nude mice were subjected to whole brain T2-weighted MRI (T2W-MRI) using a 3.0T MRI scanner (CG MUC48-H300-AG 3.0T, china). One day later, the nude mice were intravenously injected with PCN (15 mg kg) dispersed in PBS solution through the caudal cap ^-1 ). Then, 3D Photoacoustic (PA) images of the entire brain were monitored at different time points using the Vevo LAZER system (fuji film, visual system, canada). Different concentrations (0, 125, 250, 500, 1000. Mu.g.mL) were performed in PA-free tubes before PA imaging ^-1 ) PA absorption and PA imaging of PCNAnd (6) carrying out calibration. For in vivo photothermal assessment, a thermocouple probe was inserted into the intracranial glioma following exposure of the area of the skull corresponding to the area of the glioma. 808nm laser near infrared stimulator (1.0W cm) ^-2 ) The scalp was irradiated and intracranial temperatures were recorded.

6.2 results of the experiment

The uptake of PCN by intracranial tumors was monitored using Photoacoustic (PA) imaging. As shown by PA in a, b of fig. 18, intravenous PCN had time-dependent targeted accumulation in BALB/c mice carrying U87. A significant PA signal was observed at the tumor site 8 hours after i.v. injection. The location of the PA imaging is consistent with the tumor area we have previously located by MRI. After 24 hours, PA signal gradually decreased due to PCN clearance from the tumor. These results highlight that PCN not only can achieve tumor uptake by increasing permeability and detection (EPR) effects, but also has a high potential to track glioma visualization through PA imaging capabilities. We studied near-infrared induced PCN hyperthermia in vivo, recognizing that tumors accumulate highly for 8 hours. The round skull was removed during tumor inoculation to increase the penetration of NIR. We investigated 808nm laser-induced PCN hyperthermia in vivo because it has high tumor accumulation at 8 hours. The round skull was removed during tumor inoculation to increase the penetration of NIR (d of fig. 18). After exposing the area of the skull bone corresponding to the area of glioma, a thermocouple probe was inserted into the intracranial glioma (e of fig. 18). Tumor-bearing mice after intravenous injection of PCN 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 (1.0W. Cm) ^-2 ) Resulting in a temperature increase to about 50 c, while the temperature increase in the control group (normal brain tissue) was less pronounced. Accordingly, it also has power dependency and time dependency (e of fig. 18).

The ability of PCN to alleviate tumor hypoxia in vivo was studied by immunofluorescence staining for Hypoxia Inducible Factor (HIF) -1 α. Tumor hypoxia occurs in a variety of solid tumors, often resulting in resistance and metastasis of cancer cells. Compared with the green fluorescence of the control group, the fluorescence detected in the PCN-treated tumor was less, indicating that the nanosystem in TME can effectively alleviate tumor hypoxia. Notably, the PCN + NIR group showed a significant reduction in hypoxia signal, indicating that tumor hypoxia was further reduced by mild photothermal effect (fig. 18 c).

To verify the tumor growth inhibition effect on U87 tumor-bearing mice, a daily intravenous (i.v) injection concentration of 15mg kg was administered on

days

14 and 16 after tumor cell implantation ^-1 (200. Mu.L) of 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. At the same time, the intracranial tumor is reconstructed and imaged three-dimensionally using 3D slicer software to obtain a more accurate tumor size. As shown in f, j of fig. 18, tumors of the control group and PN group continued to grow over time. The PCN group was only partially tumor-inhibiting. In contrast, mice in the PCN + NIR group showed the greatest inhibitory effect. PCN + NIR treatment not only significantly prolonged survival time but also reduced weight loss compared to the other groups (g-i of fig. 18). Brain tumor tissue was collected on day 28 and then evaluated for apoptosis. Combined PCN and NIR treatment resulted in significant tumor apoptosis (k in fig. 18). Based on the above experiments, we found that free radical-based therapy has an inhibitory effect on tumors and produces better therapeutic effects in cooperation with photothermal therapy.

In addition, the skull of a human being has a certain thickness and is very hard. If one wants to generalize this treatment protocol to the clinic, it is not imaginable to remove the skull for photothermal therapy only. Clinically, it is also common for patients with glioblastoma to retain no bone flap after surgery. Based on this, the invention is designed to remove bone flap in malignant glioma operation, PCN material can combine with the hemostatic substrate (Surgiflo) commonly used by neurosurgeons to Surgiflo @ PCN, then is applied to the surface of tumor cavity after operation, which is more convenient for later treatment.

7ICD marker assay and DC maturation

CDT and mild PTT are critical for the induction of ICD in dead cancer cells, which will synergistically increase oxidative stress in tumors. To investigate whether PCN is able to induce ICD, the present invention determined several DAMPs during ICD by incubating GL261 cells with PCN. In-mold GL261 cells with PCNPreincubation in pseudo-TME followed by mild near-infrared laser (0.8W cm) ^-2 5 min) to maintain the temperature of the cell culture medium at about 45 ℃. As a result, it was found that the PCN group and the PCN + NIR group significantly induced Calreticulin (CRT) exposure in tumor cells within 6 hours, compared to the control group and the NIR group. Flow cytometry analysis showed that of these dead cells, CRT exposure was highest in the PCN + NIR treated group, with an efficiency of about 4.68 times that of the control group (f, g of fig. 19). The extracellular ATP release had the same trend (h of fig. 19). After 24 hours, the highest concentration of high mobility group box protein B1 (HMGB 1) was detected in the PCN + NIR group (fig. 19, i). At mildly elevated temperatures, PCN-induced ICD was enhanced. In other words, PCNs with multi-enzyme activity induce ICD under the control of NIR. The present invention recognizes that PCN with multi-enzyme activity can generate ROS, which will synergistically increase oxidative stress in tumors. To confirm our hypothesis, their performance in modulating intracellular oxidative stress was investigated. As shown in c of fig. 20, the PCN-treated cells showed higher intensity of DCF intracellular fluorescence compared to the control group. After near infrared radiation, the intracellular fluorescence is further enhanced. FIG. 20 c is a graph showing the results of simulation of TME in the presence or absence of laser irradiation (808nm, 0.8w.cm) ^-2 ) In the case of (1), GL261 cells and PCN (100. Mu.g. Multidot.mL) ^-1 ) Relative survival after incubation; these data suggest that we induce ROS production through PCN multienzyme activity and further enhance and coordinate this process through PTT.

Dendritic Cells (DCs) play a central role in activating the immune system, being responsible for activating native T cells. To mimic these processes, we investigated PCN-induced immunogenicity by assessing the maturity of bone marrow-derived dendritic cells (BMDCs) in the transwell system. GL261 tumor cells were seeded in the upper chamber, treated with PCN and/or NIR, and DC cells were seeded in the lower chamber. CD86 and CD80 staining further confirmed DC maturation, which is a typical marker for the surface of mature DCs. As shown in j, k of fig. 19, the percentage of mature DCs in the PCN group and the PCN + NIR group were significantly increased 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.

Effect of 8Surgiflo @ PCN on in vivo gliomas

Inspired by the excellent enzyme mimetic activity and immune activation properties of PCN, the in vivo antitumor properties in the surgical resection model were next evaluated. Surgiflo is commonly used by neurosurgeons and has a multi-spatial structure that can be fully absorbed by the brain (fig. 19 b). We have found that a mixture of surgiflo and PCN (surgiflo @ PCN) not only creates a variety of shapes for easy application to the tumor cavity (a of FIG. 19) post-operatively, but also provides stable release of PCN. Flow cytometry analysis showed that the release and cellular uptake rates of PCN were stably time-dependent in the co-culture system (c of fig. 19). The PD-1 antibody (α PD-1) is useful for inhibiting tumor immune evasion as a checkpoint inhibitor approved by the Food and Drug Administration (FDA).

A glioma surgical resection model was constructed (a of fig. 21). First, stable luciferase-transfected GL261 (Luci + GL 261) cells were intracranially injected into mice to generate mouse orthotopic glioma models. On day 14 after implantation, mice with similar fluorescence intensity were screened by bioluminescence. Mice were randomized into groups prior to surgery. Mice were divided into six groups (6 per group): (1) control group, (2) α PD-1 group, (3) Surgiflo @ PCN, (4) Surgiflo @ PCN + α PD-1, (5) Surgiflo @ PCN + NIR, (6) Surgiflo @ PCN + NIR + α PD-1. Surgiflo @ PCN for the Surgiflo @ PCN treatment groups (3) - (6) (dosage of PCN 0.75 mg-kg) ^-1 Surgiflo 7.5 μ L kg ^-1 ) The tumor resection cavity of each mouse was tiled. At 8 hours after tiling, tumors of mice in groups (5) and (6) received 808nm laser irradiation (0.8W. Cm) ^-2 ) For 5 minutes. On the 14 th, 17 th and 20 th days after the injection, α PD-1 was intravenously injected to the mice at a dose of 0.75 mg/kg ^-1 . The operation process is shown as a in fig. 21. Glioma-bearing mice were able to tolerate surgical procedures and had no nerve damage after surgery (b of fig. 21 is the procedure of surgical removal of the tumor from the brain of mice bearing GL261, b1, image of the tumor (purple arrow) formed in the mouse brain 14 days after implantation b2, microsurgical removal of the tumor from the brain, GBM cavity (black arrow), bone window (red arrow);b3, surgiflo @ PCN in the cavity; b4, tumor resection). The thermocouple probe was inserted into the resection cavity and used 6 hours later with surgiflo @ pcn (fig. 21 c). The internal temperature of the tumor was then recorded. In fact, 5 minutes of irradiation (0.8W cm) ^-2 ) Resulting in a temperature rise to about 45 deg.c. Accordingly, it also has power dependency and time dependency (d of fig. 21). Using a 808nm laser (0.8W cm) ^-2 5 min) NIR irradiation was performed on mice treated 6 hours post-surgery in the pattern shown in a of fig. 21 to achieve mild PTT. The next day, bioluminescence analysis further eliminated the disqualified mice to ensure similar tumor remains. Bioluminescence is used to continuously monitor tumor growth. As shown by the bioluminescence assay, catalytic immunotherapy with or without mild PTT showed effective inhibition of tumor recurrence. Tumor volume increase significantly slower in the surgiflo @ PCN group than in the control group or alpha PD-1 group alone (P)<0.01 Notably the Surgiflo @ PCN + NIR + alpha PD-1 group has the strongest inhibitory effect on tumor regeneration (P)<0.001 The group of mice showed no even significant tumors at the end of the study (e, g of figure 21). Furthermore the surgiflo @ pcn + NIR + α PD-1 group significantly extended the lifespan of tumor bearing mice, reducing the risk of tumor recurrence (figure 21 f). The Surgiflo @ PCN + alpha PD-1 group has no significant difference from the control group in the aspects of prolonging survival time and inhibiting tumors, and the Surgiflo @ PCN + alpha PD-1 group does have better curative effect. Histological observations of tumor-bearing brains were collected 28 days post-implantation in the surgiflo @ pcn + NIR + alpha PD-1 group. For brain slicing H&And E, dyeing. A small residual Surgiflo was found in the tumor cavity. Some post-operative residual tumor was found around the tumor cavity. In further TUNEL staining, extensive apoptotic cells were found to be detected not only at the residual tumor sites, but also in tumor satellite 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 some of the tested mice (fig. 22, a is a schematic diagram of experimental design). For the mouse surgiflo @ pcn + NIR + α PD-1 group in the group, the numbers of CD8+ Cytotoxic T Lymphocytes (CTL) and CD4+ helper T lymphocytes were significantly greater than the control group (b, d of fig. 22 and fig. 23), indicating effective activation of the anti-tumor immune response. To further demonstrate that sub-hypoxic and retro-hypoxic environments can reprogram immunosuppressive TMEs, regulatory T cells (Tregs, CD3+ CD4+ Foxp3 +) were studied. As shown in c, e of fig. 22, treg frequency was significantly reduced to 4.15 ± 0.26% under normal conditions, and Treg frequency was significantly lower in surgiflo @ pcn and surgiflo @ pcn + α PD-1 groups than in the control group. Surgiflo @ pcn + NIR and surgiflo @ pcn + NIR + α PD-1 (fig. 22 f) were monitored for significant increases in CTL to Treg cell ratios, suggesting that they can reprogram immunosuppressive TME, inducing expression of immune cells with anti-tumor activity. Furthermore the surgiflo @ pcn + NIR + α PD-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 hyperthermia-induced oxidative stress and activation of ICDs can successfully reprogram TMEs, ultimately inhibiting the regeneration of post-operative GBMs.

9 in vivo biosafety assessment

Mice were injected intravenously with a 30mg/kg dose of PCN. At 14 days post-implantation, mice were sacrificed and blood was collected to obtain blood routine and serological chemical data. 14 days after implantation, major organs including heart, liver, spleen, lung and kidney were removed and H was performed&And E, dyeing. The present inventors investigated the effect of PCN on erythrocytes. The hemolysis results showed that even concentrations as high as 0.2 mg/mL in PBS were present ^-1 Hemolysis was also negligible, indicating good PCN biocompatibility (fig. 24). Secondly, in vivo blood biochemical and blood routine analyses, particularly liver and kidney function marker analyses, showed that even 30mg kg of intravenous injection was administered over 2 weeks ^-1 Also, there was no significant liver/kidney toxicity and blood toxicity for PCN (AST, ALT, BUN, WBC, RBC, HCT, HGB, PLT in order, fig. 25 a-h). Third, hematoxylin and eosin (H) were also performed on major organ tissues of 4-week-sacrificed mice&E) Staining (fig. 26). No detectable signs of damaged tissue were observed. All these results firmly indicate that intravenous PCN has negligible systemic toxicity, representing a safe approach to the tumor treatment platform.

Conclusion 10

In the clinic, treatment of GBM remains largely dependent on standard surgical resection. However, due to the infiltrative nature of gliomas, surgical resection does not completely remove tumor cells, making the rate of tumor recurrence extremely high. Therefore, it is important to inhibit the growth of residual infiltrating glioma cells after GBM excision. Herein, the present invention proposes a novel artificial enzyme PCN having enzyme mimetic activities (including POD-like activities CAT and OXD-like activities) for use in catalytic immunotherapy. PCN can reprogram immunosuppressive TME and eliminate residual tumors by catalyzing the production of reactive oxygen species to gradually increase oxidative stress in tumors and synergistically induce tumor immunogenic death with mild PTT. The present invention combines the hemostatic matrix system commonly used by neurosurgeons (surgiflo) with PCN (surgiflo @ PCN) to make it possible to treat the infiltrates of gliomas during surgery. This design has the following advantages:

(1) Surgiflo @ PCN the PCN is applied to the inside of a tumor cavity in situ without changing the inherent characteristics of the surgiflo, so that the concentration and the acting time of the PCN are greatly increased. (2) The efficiency of mild photothermal therapy can be conveniently improved after removal of a small "bone window" (which also corresponds to post-clinical treatment). (3) Surgiflo is a mature hemostatic material that can significantly reduce postoperative bleeding and other complications in clinical applications, particularly in microneurosurgery. This strategy will provide new anti-tumor immunity opportunities for patients undergoing brain tumor resection, inhibiting post-operative tumor recurrence, which may translate in the future into the clinical use of artificial enzymes for biomedical applications.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims

1. A preparation method of a composite material for inhibiting postoperative tumor recurrence is characterized by comprising the following steps:

2. The preparation method according to claim 1, wherein the preparation method specifically comprises: dissolving 10-15mg of polyvinylpyrrolidone in 1-2mL of deionized water to obtain a PVP solution, then adding a mixed solution of copper chloride and palladium (II) potassium chloride into the PVP solution, stirring at 25-28 ℃ for 1-2min, then adding 1-1.5mL of ascorbic acid, carrying out ultrasonic treatment for 15-20min, and dialyzing with deionized water through a dialysis bag with molecular weight cutoff of 10-15KDa for 2-3 days to obtain the composite material for inhibiting postoperative tumor recurrence;

3. A composite material for inhibiting postoperative tumor recurrence obtained by the production method according to any one of claims 1 to 2.

4. Use of the composite material according to claim 3 for the preparation of a medical material for inhibiting postoperative tumor recurrence.

5. The use according to claim 4, wherein the composite material in combination with an absorbable hemostatic fluid gelatin Surgiflo is effective in inhibiting post-operative tumor recurrence.

6. The use according to claim 5, wherein the composite material is injected into a patient in combination with absorbable hemostatic fluid gelatin Surgiflo, and activated oxygen is generated by chemo-kinetic therapy and photothermal therapy to enhance oxidative stress of tumor cells, thereby killing tumor cells and inhibiting postoperative tumor recurrence.

7. The use according to claim 5, wherein the composite material is injected into a patient in combination with the absorbable hemostatic fluid gelatin Surgiflo, to induce immunogenic cell death, reverse the immunosuppressive tumor microenvironment, enhance the anti-tumor immune response, to inhibit postoperative tumor recurrence.

8. A medical material for inhibiting postoperative tumor recurrence comprising the composite material of claim 3 or the composite material of claim 5 in combination with a absorbable hemostatic fluid gelatin Surgiflo-derived material.

9. The medical material according to claim 8, wherein the tumor comprises a glioma.