Fe (III)-EGCG (epigallocatechin gallate) network coated copper sulfide nano-composite and application thereof

Abstract

The invention discloses a Fe (III)-EGCG (epigallocatechin gallate) network coated copper sulfide nano-composite and application thereof, and belongs to the technical field of biological medicines. Polyethyleneimine is used as a nano-carrier, PEG-FA and FI are modified on the surface of the nano-carrier, CuS NPs is synthesized in situ in an internal cavity of the nano-carrier, residual amino groups on the surface of PEI.NH2 are functionalized through acetylation, and finally, FA-CuS PENs are coated with a metal polyphenol network Fe-EGCG, so that the nano-carrier is obtained. The Fe (III)-EGCG network coated copper sulfide nano-composite with the cancer cell MR imaging and chemical / photo-thermal / chemical kinetics combined treatment function is constructed. The nano-composite disclosed by the invention can realize high-amount uptake of cancer cells highly expressed by an FA receptor, and further realizes targeted cancer cell MR imaging and chemical / photo-thermal / chemical kinetics combined therapy.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a Fe(III)-EGCG network-wrapped copper sulfide nanocomposite and application thereof. BACKGROUND

[0002] At present, there are many treatment methods for cancer, including surgical resection, chemotherapy, radiotherapy, photothermal therapy (PTT), chemical dynamics therapy (CDT), photodynamic therapy (PDT) and immunotherapy. Surgical resection is the main way to treat tumors, but it has the disadvantages of large side effects, high recurrence rate and the like, resulting in poor tumor treatment effect and prognosis. Therefore, new tumor treatment methods have become a hot direction. PTT relies on materials with photothermal effect to convert light energy into heat energy, and kills cancer cells through high temperature. Based on the fact that normal cells have higher heat resistance than cancer cells, moderate high temperature can kill cancer cells while avoiding toxic side effects on normal cells (Hirsch, L.R.; Stafford, R.J. et al. Proc Natl Acad Sci USA, 2003, 100(23): 13549-13554). With the rapid development of nanotechnology, copper sulfide nanoparticles (CuS NPs) stand out due to their excellent optical properties. They are easy to synthesize, have high absorbance in the near-infrared (NIR) region, good photothermal conversion coefficient and light stability, and can enhance the contrast of photoacoustic imaging, making them widely used in imaging and PTT of tumors (Sun, L.; Fu, X. et al. ACS Appl Mater Interfaces, 2020, 12(41): 46146-46161). However, CuS NPs-mediated PTT also has its own limitations, such as low effective dose of CuS NPs after delivery to tumor sites by the body circulation, which limits its PTT effect. Therefore, it is urgent to develop a strategy to improve the enrichment of CuS NPs system in tumor sites.

[0003] Currently, common strategies to improve the enrichment of nanomaterials in tumor sites include: 1. Regulating nanoscale to enhance the penetration and retention (EPR) effect; 2. Modifying targeting molecules to take advantage of the specificity of target ligand-receptor binding; 3. Modifying zwitterions to impart charge reversal properties. Among them, target ligand-receptor binding is a common means, so targeting molecules can be modified on CuS NPs system to improve their enrichment in cancer cells, thereby improving their mediated tumor PTT treatment efficiency.

[0004] In addition, after laser irradiation of the tumor site, the CuS NPs-mediated PTT is limited by the depth of laser penetration, which affects the photothermal treatment effect of the tumor. This superficial administration of photothermal therapy (S-PTT) results in its penetration through the tissue depth being superficial, and this method is only limited to the treatment of superficial tumors, making it impossible to comprehensively and deeply treat deep tumor tissues, leaving untreated active tumor cells and causing tumor recurrence (Sun, L.; Bai, H. et al. Colloids Surf B, 2022, 214: 112462). Therefore, although the tumor PPT treatment method has its unique advantages, it also has its own defects, making it difficult to comprehensively and deeply treat tumors, and the tumor treatment efficiency is poor.

[0005] As a new tumor treatment method, CDT has tumor treatment specificity. Based on free radicals, it uses the high amount of hydrogen peroxide (H2O2) in the tumor microenvironment (TME) to generate hydroxyl radicals (·OH) through Fenton / Fenton-like reactions, thereby killing tumor cells. At the same time, the extremely low amount of H2O2 in normal cells cannot induce the generation of ·OH, so CDT has tumor specificity (Zang, C.; Bu, W. B. et al. Angew Chem Int Ed, 2016, 55(6): 2101-2106). In addition, it is not affected by external triggering factors, is beneficial to the treatment of deep tissues, has small side effects, and can make up for the influence of the limited treatment effect of PTT by the depth of laser penetration. The commonly used CDT metal elements include: iron (Fe), copper (Cu), manganese (Mn), titanium (Ti), nickel (Ni), cobalt (Co), etc. Among them, iron ions (Fe(III)) can participate in Fenton reactions, and due to its high oxidation capacity and mild reaction conditions, it is widely used in multiple biomedical fields (Sun, Y.; Zhou, P. et al. Chem Eng J, 2022, 450(4): 138423; Wu, C. H.; Dong, C. D. et al. React Kinet Mech Catal, 2022, 135(1): 441-457). Therefore, in order to further solve the problem of poor tumor treatment efficiency of CuS NPs-mediated PTT, CuS NPs can be combined with Fe(III) to construct a PTT / CDT combined treatment system to improve the tumor treatment efficiency.

[0006] In order to make up for the low enrichment degree of the CuS NPs system at the tumor site and the poor effect of the tumor PTT mediated by itself, the application constructs a novel CuS NPs nanocomposite, combines the targeting molecule FA, CuS NPs and Fe(III) organically, and attempts to realize efficient tumor diagnosis and treatment by using the targeting strategy and the PTT / CDT combined treatment strategy through the construction of the multifunctional nanocomposite. SUMMARY

[0007] One of the purposes of the application is to provide a Fe(III)-EGCG network wrapped copper sulfide nanocomposite, which is prepared by the following steps:

[0008] Step 1, adding (3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to a dimethyl sulfoxide solution of folic acid (FA) for activation, and then adding the solution to a dimethyl sulfoxide solution of amino-polyethylene glycol-carboxyl (NH2-PEG-COOH, Mw=2000) for reaction to obtain FA-PEG-COOH;

[0009] Step 2, adding (3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to an aqueous solution of FA-PEG-COOH for activation, and then adding to an aqueous solution of polyethyleneimine (PEI.NH2) for reaction to obtain PEI.NH2-(PEG-FA);

[0010] Step 3, adding a dimethyl sulfoxide solution of fluorescein isothiocyanate (FI) to a dimethyl sulfoxide solution of PEI.NH2-(PEG-FA) for reaction under dark and stirring to obtain PEI.NH2-(PEG-FA)-FI;

[0011] Step 4, adding an aqueous solution of copper chloride dihydrate (CuCl2·2H2O) to an aqueous solution of PEI.NH2-(PEG-FA)-FI, stirring under dark, then adding an aqueous solution of sodium sulfide nonahydrate (Na2S·9H2O) under heating, stirring under dark, after the temperature of the mixed solution decreases to room temperature, adding triethylamine (N(C2H5)3) for reaction under dark, and then adding acetic anhydride (Ac2O) for reaction under dark to obtain FA-CuS PENs;

[0012] Step 5, adding an aqueous solution of iron chloride (FeCl3) to an aqueous solution of FA-CuS PENs, stirring to mix, then adding an aqueous solution of epigallocatechin gallate (EGCG), and stirring for reaction to obtain the Fe(III)-EGCG network wrapped copper sulfide nanocomposite FA-CuS PENs@Fe-EGCG.

[0013] Further, in step 1, the molar ratio of EDC, NHS and FA is 1-1.5:1-1.5:1, and the molar ratio of FA and NH2-PEG-COOH is 1.2-1.5:1.

[0014] Further, in step 2, the molar ratio of EDC, NHS and FA-PEG-COOH is 10-12:10-12:1, and the molar ratio of FA-PEG-COOH and PEI.NH2 is 20-25:1.

[0015] Further, in step 3, the molar ratio of FI and PEI.NH2-(PEG-FA) is 3-5:1.

[0016] Further, in step 4, the molar ratio of CuCl2·2H2O and PEI.NH2-(PEG-FA)-FI is 50-52:1, the molar ratio of Na2S·9H2O and CuCl2·2H2O is 3:1, and the molar ratio of N(C2H5)3, Ac2O and PEI.NH2-(PEG-FA)-FI is 120-660:100-550:1.

[0017] Further, in step 4, the heating temperature is 70℃.

[0018] Further, in step 5, the molar ratio of FeCl3 and FA-CuS PENs is 10-12:1, and the molar ratio of EGCG and FeCl3 is 2:1.

[0019] The second object of the present application is to provide the use of the above Fe(III)-EGCG network wrapped copper sulfide nanocomposite in the preparation of tumor diagnosis and treatment products.

[0020] Folic acid (FA) as a targeting ligand can specifically bind to cancer cells with high expression of folate receptor. First, the present application modifies FA on the surface of hyperbranched polyethyleneimine to achieve its targeting of cancer cells with high expression of FA receptor, so as to achieve the purpose of specific delivery of FA-CuS PENs@Fe-EGCG to cancer cells; at the same time, the present application uses polyethylene glycol (PEG) to enhance the biocompatibility of the nanocomposite, prolong the in vivo circulation time, and increase the stability of the functionalized hyperbranched polyethyleneimine wrapped CuSNPs; the present application modifies FI on the surface of hyperbranched polyethyleneimine, which can act as a fluorescent tracer. Then, the present application uses CuCl2·2H2O and Na2S·9H2O to in situ synthesize CuS NPs in the hydrophobic cavity of the functionalized hyperbranched polyethyleneimine at 70 DEG C, so as to endow the nanocomposite with the function of photothermal therapy of cancer cells. In addition, the present application acetylates the remaining amino groups on the surface of the functionalized hyperbranched polyethyleneimine, which reduces the surface potential and improves the biocompatibility of the nanocomposite. Finally, the present application coats the Fe(III)-EGCG network on the outside of FA-CuS PENs, which can endow the nanocomposite with the ability of MR imaging, chemotherapy and chemical kinetic therapy of cancer cells at the same time.

[0021] Advantages

[0022] (1) The FA-CuS PENs@Fe-EGCG prepared by the present application has a simple synthesis method and is easy to operate, and has good biomedical application value.

[0023] (2) The modification of FA in the present application can improve the uptake of FA-CuS PENs@Fe-EGCG by cancer cells with high expression of FA receptor; Fe(III) can not only mediate T1 weighted MR imaging, but also mediate CDT mediated by Fe(III), EGCG mediated chemotherapy and CuSNPs mediated PTT, which can advantageously complement and synergistically enhance the treatment efficiency of cancer cells. The metal polyphenol network (MPNs) shell formed by the complexation of Fe(III) and EGCG coated on the surface of FA modified CuS NPs can effectively integrate FA, CuS NPs, EGCG and Fe(III), so that FA-CuS PENs@Fe-EGCG is specifically taken up by cancer cells with high expression of FA receptor, and MR imaging, chemotherapy / photothermal / chemical kinetic combined therapy of targeted cancer cells is realized. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The synthesis of FA-CuS PENs@Fe-EGCG prepared by the present application is shown in the figure.

[0025] Figure 2UV-Vis spectra of (a) FA-PEG-COOH, (b) PEI.NH2-(PEG-FA), (c) PEI.NH2-(PEG-FA)-FI, (d) PEI.NH2-mPEG-FI, and (e) PEI.NH2-mPEG-FI prepared in the present application dispersed in deuterium water (D2O) 1 H NMR spectra.

[0026] Figure 3 UV-Vis spectra of (a) FA-PEG-COOH, PEI.NH2-(PEG-FA), PEI.NH2-(PEG-FA)-FI, PEI.NH2-mPEG-FI, (b) FA-CuS PENs, CuS PENs, and (c) FA-CuS PENs@Fe-EGCG, FA-CuS PENs@EGCG, Fe-EGCG, EGCG prepared in the present application dispersed in water.

[0027] Figure 4 (a) TEM image and (b) high-resolution TEM image of FA-CuS PENs@Fe-EGCG, (c) TEM image of FA-CuS PENs@EGCG, (d) TEM image of FA-CuS PENs@Fe-EGCG dissolved in pH = 6.5 buffer solution, (e) size distribution histogram of FA-CuS PENs@Fe-EGCG, and (f) FA-CuS PENs@EGCG prepared in the present application.

[0028] Figure 5 (a) XPS survey spectrum of FA-CuS PENs@Fe-EGCG, and (b) Cu 2p and (c) Fe 2p XPS spectra of FA-CuS PENs@Fe-EGCG with high resolution of FA-CuS PENs@Fe-EGCG prepared in the present application.

[0029] Figure 6 Photothermal properties and photothermal stability test of FA-CuS PENs@Fe-EGCG prepared in the present application. The near-infrared laser (1064 nm, 1.0 W / cm2) was used to irradiate the sample for 10 min, and the temperature of the sample was measured by a thermal infrared camera. 2(a) Temperature rise curves and (b) temperature changes (ΔT) of FA-CuS PENs@Fe-EGCG aqueous solutions with different Cu concentrations under irradiation. (c) Temperature rise curves of FA-CuS PENs@Fe-EGCG aqueous solutions irradiated with near-infrared lasers of different powers ([Cu]=0.6mM). (d) Temperature changes over time of FA-CuS PENs@Fe-EGCG aqueous solutions under near-infrared laser irradiation heating / cooling for five cycles and (e) one cycle. (f) Linear fitting relationship between the natural cooling time and -lnθ of FA-CuS PENs@Fe-EGCG aqueous solutions.

[0030] Figure 7 With / without laser (1064nm, 1.0W / cm) 2 (a) Fe(III) and (b) EGCG release curves of FA-CuS PENs@Fe-EGCG under different pH conditions of irradiation.

[0031] Figure 8 The (a) MB solution prepared for this invention was reacted with FA-CuS PENs@Fe-EGCG+H2O2, FA-CuS PENs@Fe-EGCG+H2O2+GSH, and FA-CuS PENs@Fe-EGCG+H2O2+laser (1064nm, 1.0W / cm) respectively. 2 (a) UV-Vis spectrum of FA-CuS PENs@Fe-EGCG+H2O2+MB solution after incubation for 6 h (5 min). (b) UV-Vis spectrum of FA-CuS PENs@Fe-EGCG+H2O2+MB solution at different time points. (c) Residual percentage of MB in FA-CuS PENs@Fe-EGCG+H2O2+MB solution over time under different pH conditions. (d) UV-Vis spectrum of FeCl3 solution after incubation with H2O2+MB and EGCG+H2O2+MB for 12 h.

[0032] Figure 9 (a) T1-weighted MR pseudocolor images of FA-CuS PENs@Fe-EGCG with different Fe concentrations and (b) linear fitting plot of 1 / T1 as a function of Fe concentration in FA-CuS PENs@Fe-EGCG.

[0033] Figure 10 The hydration particle size curves of (a) FA-CuS PENs@Fe-EGCG, FA-CuS PENs@EGCG, and CuS PENs@Fe-EGCG aqueous solutions prepared for this invention as a function of time, and (b) FA-CuS PENs@Fe-EGCG aqueous solution under a 1064 nm laser (1.0 W / cm²) are shown. 2UV-Vis spectra before and after irradiation (t = 5 min).

[0034] Figure 11 Pictures of hemolysis rate of red blood cells after 2 h treatment of FA-CuS PENs@Fe-EGCG with different Fe concentrations. Material A is FA-CuS PENs@Fe-EGCG ([Fe] = 100 mM) solution without red blood cells.

[0035] Figure 12 Flow cytometry test results of (a) 4T1-HFAR cells treated with PBS for 4 h, (b) 4T1-LFAR cells and (c) 4T1-HFAR cells treated with CuS PENs@Fe-EGCG for 4 h, and (d) 4T1-LFAR cells and (e) 4T1-HFAR cells treated with FA-CuS PENs@Fe-EGCG for 4 h.

[0036] Figure 13 Flow cytometry test results of (a) mean fluorescence intensity and (b) cellular uptake rate of 4T1-HFAR and 4T1-LFAR cells treated with different materials for 4 h, respectively.

[0037] Figure 14 CLSM images of 4T1-HFAR cells treated with PBS for 4 h, 4T1-LFAR cells and 4T1-HFAR cells treated with CuS PENs@Fe-EGCG and FA-CuS PENs@Fe-EGCG for 4 h, respectively.

[0038] Figure 15 ICP-OES test results of 4T1-LFAR and 4T1-HFAR cells incubated with FA-CuS PENs@Fe-EGCG with different Fe concentrations for 4 h, respectively.

[0039] Figure 16 (a) T1-weighted MR imaging pseudo-color images and (b) corresponding MR signal values of 4T1 cells incubated with (1) FA-CuS PENs@Fe-EGCG and (2) CuS PENs@Fe-EGCG with different Fe concentrations for 4 h.

[0040] Figure 17(a) Cell viability of L929 cells after incubation with FA-CuS PENs@Fe-EGCG, CuS PENs@Fe-EGCG and FA-CuS PENs@EGCG with different CuS concentrations for 24 h, respectively. (b) Cell viability of 4T1 cells after incubation with CuS PENs, FA-CuS PENs, FA-CuS PENs@EGCG, CuS PENs@Fe-EGCG and FA-CuS PENs@Fe-EGCG with different CuS concentrations for 24 h, respectively. (c) Cell viability of 4T1 cells after treatment with different materials ([CuS]=0.32 mM) with / without laser (1064 nm, 1.0 W / cm 2 , 5 min) irradiation. 2 (d) Cell viability of 4T1 cells after treatment with different materials ([CuS]=0.32 mM) with laser (1064 nm, 1.0 W / cm 1 , 5 min) irradiation.

[0041] Figure 18 (a) Annexin V-FITC / PI double staining test chart and (b) percentage of apoptotic cells of 4T1 cells after incubation with different materials for 24 h. (c) ROS characterization chart and (d) relative ROS level quantification results, (e) GSH content, (f) LPO characterization chart and (g) relative LPO level quantification results of 4T1 cells after treatment with different materials for 6 h. DETAILED DESCRIPTION

[0042] The preferred embodiments of the present application will be described in detail hereinafter with reference to the embodiments. It is to be understood that the following embodiments are given by way of illustration only and are not intended to limit the scope of the present application. Various modifications and substitutions can be made by those skilled in the art without departing from the spirit and scope of the present application.

[0043] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0044] The materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified.

[0045] The present application uses nuclear magnetic resonance hydrogen spectrum (1H-NMR) to analyze the chemical structure of the product. 1The prepared FA-CuS PENs@Fe-EGCG is subjected to basic characterization by means of H NMR, ultraviolet-visible absorption spectrum (UV-Vis), transmission electron microscopy (TEM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy (XPS), in vitro photothermal performance test, T1 relaxation rate test and the like test means; the targeting cancer cell capability, in vitro cancer cell combined treatment effect, targeted cancer cell MR imaging, ROS production capability, GSH consumption capability, LPO induction production capability, cancer cell apoptosis, blood compatibility and the like of the FA-CuS PENs@Fe-EGCG are evaluated by means of flow cytometry, laser scanning confocal microscopy (CLSM), inductively coupled plasma emission spectroscopy (ICP-OES), cell proliferation-toxicity (CCK-8) test method, cell MR imaging analysis, intracellular reactive oxygen species (ROS) analysis, intracellular glutathione (GSH) analysis, intracellular lipid peroxide (LPO) analysis, cell apoptosis test and hemolysis.

[0046] Example 1

[0047] Step 1, 100 mg of NH2-PEG-COOH (Mw = 2000) is weighed and dissolved in 15 mL of DMSO, 33.10 mg of FA is dissolved in 10 mL of DMSO, 14.4 mg of EDC is dissolved in 5 mL of DMSO, and 8.6 mg of NHS is dissolved in 3 mL of DMSO. First, the FA and EDC solution are mixed and stirred for 3-4 min, then the NHS solution is added to the above solution and stirred for 3 h, finally the mixed solution of FA, EDC and NHS is added dropwise to the NH2-PEG-COOH solution and stirred for 3 days. After the reaction is completed, the above solution is dialyzed in ultrapure water in a dialysis bag with a molecular weight cut-off of 1000 for 3 days (3 times / day, 2 L / time), and then freeze-dried to obtain the FA-PEG-COOH material. The molar ratio of NH2-PEG-COOH, FA, EDC and NHS is 1:1.5:1.5:1.5.

[0048] Step 2, 22.5 mg of PEI.NH2(Mw=25000) was weighed into 10 mL of ultrapure water, 43 mg of FA-PEG-COOH was dissolved in 10 mL of ultrapure water, 34.5 mg of EDC was dissolved in 10 mL of ultrapure water, and 20.7 mg of NHS was dissolved in 5 mL of ultrapure water. First, the EDC solution was added to the FA-PEG-COOH solution and stirred for 3-4 min, then the NHS solution was added to the above solution and stirred for 3 h, and finally the mixed solution of FA-PEG-COOH, EDC and NHS was added dropwise to the PEI.NH2solution and stirred for 3 days. After the reaction was completed, the above solution was dialyzed in ultrapure water for 3 days (3 times / day, 2 L / time) using a dialysis bag with a molecular weight cut-off of 8000-14000, and then freeze-dried to obtain PEI.NH2-(PEG-FA). The molar ratio of PEI.NH2, FA-PEG-COOH, EDC and NHS was 1:20:200:200.

[0049] Step 3, 60 mg of PEI.NH2-(PEG-FA) was dissolved in 13 mL of DMSO, and 2.0 mg of FI was dissolved in 2 mL of DMSO. The FI solution was added dropwise to the PEI.NH2-(PEG-FA) solution and stirred in the dark for 24 h. After the reaction was completed, the above solution was dialyzed in ultrapure water for 3 days (3 times / day, 2 L / time) using a dialysis bag with a molecular weight cut-off of 8000-14000, and then freeze-dried to obtain PEI.NH2-(PEG-FA)-FI. The molar ratio of PEI.NH2-(PEG-FA) and FI was 1:5.

[0050] Step 4, 43 mg of PEI.NH2-(PEG-FA)-FI was dissolved in 10 mL of ultrapure water, 6.4 mg of CuCl2·2H2O was dissolved in 5 mL of ultrapure water, 27.0 mg of Na2S·9H2O was dissolved in 10 mL of ultrapure water. The CuCl2 solution was added dropwise to the PEI.NH2-(PEG-FA)-FI solution, stirred in the dark for 15 min, then the Na2S solution was added dropwise to the above solution under the condition of heating at 70°C, stirred in the dark for 30 min, forming a dark green solution. After the temperature of the above solution dropped to room temperature, 79 μL of N(C2H5)3 was added, stirred in the dark for 30 min, then 52 μL of Ac2O was added, and the reaction was continued in the dark for 24 h. After the reaction was completed, the above solution was dialyzed in ultrapure water for 3 days (3 times / day, 2 L / time) by selecting a dialysis bag with a molecular weight cutoff of 8000-14000, and then freeze-dried to obtain FA-CuS PENs. The molar ratio of PEI.NH2-(PEG-FA)-FI, CuCl2·2H2O, Na2S·9H2O was 1:50:150, and the molar ratio of PEI.NH2-(PEG-FA)-FI, N(C2H5)3, Ac2O was 1:500:500.

[0051] Step 5, 15 mg of FA-CuS PENs was dissolved in 5 mL of ultrapure water, then 308 μL of 8 mM FeCl3 solution was added to the FA-CuS PENs solution, vortexed, then 612 μL of 8 mM EGCG solution was added, stirred in the dark for 24 h, after the reaction was completed, the above solution was ultrafiltration centrifugation (8000 rpm, 10 min) by selecting an ultrafiltration centrifuge tube with a molecular weight cutoff of 3000, and then the product solution was obtained after ultrafiltration purification and freeze-dried to obtain FA-CuS PENs@Fe-EGCG nanocomposite. The molar ratio of FA-CuS PENs, FeCl3, EGCG was 1:10:20.

[0052] The reaction scheme is shown in the description Figure 1 .

[0053] The intermediate products in the above synthesis process were characterized by 1 H NMR, and the 1The characteristic peaks in the H NMR spectrum were assigned (qualitative analysis) and integrated (quantitative characterization), proving that the intermediates: FA-PEG-COOH, PEI.NH2-(PEG-FA), PEI.NH2-(PEG-FA)-FI were successfully synthesized. Then, the characteristic peaks were integrated and calculated to find that 0.7 FAs were modified in each molecule of FA-PEG-COOH, 13.9 FA-PEG-COOH, 9.7 FAs and 2.6 FIs were modified on the surface of each molecule of PEI.NH2-(PEG-FA)-FI, see the attached Figure 2 UV-Vis spectra of FA-PEG-COOH, PEI.NH2-(PEG-FA), PEI.NH2-(PEG-FA)-FI were scanned, see the attached Figure 3 UV-Vis spectra of FA-PEG-COOH, PEI.NH2-(PEG-FA), PEI.NH2-(PEG-FA)-FI were scanned, see the attached Figure 3 a) After PEI.NH2-(PEG-FA)-FI encapsulated CuS NPs and acetylation, the product FA-CuS PENs was formed, and its UV-Vis characterization was performed, and an absorption peak was found at 1000-1100 nm, which can be attributed to the characteristic peak of CuS NPs, thereby proving that CuS NPs were successfully encapsulated in the functionalized polyethyleneimine Figure 3 b) Compared with the ultraviolet-visible characteristic absorption peak of free EGCG at 273 nm, Fe-EGCG, FA-CuS PENs@Fe-EGCG and FA-CuS PENs@EGCG all appeared the characteristic absorption peak at 273 nm, indicating that EGCG was successfully loaded on FA-CuS PENs Figure 3 c).

[0054] Comparative Example 1

[0055] (1) Step 1, 50 mg PEI.NH2 was dissolved in 3 mL ultrapure water, 80 mg mPEG-COOH (Mw=2000) was dissolved in 3 mL ultrapure water, 76.7 mg EDC was dissolved in 5 mL ultrapure water, 46 mg NHS was dissolved in 3 mL ultrapure water. First, the EDC solution was added to the mPEG-COOH solution and stirred for 3-4 min, then the NHS solution was added to the above solution and stirred for 3 h, finally the mixed solution of mPEG-COOH, EDC and NHS was added dropwise to the PEI.NH2 solution and stirred for 3 days. After the reaction was completed, the above solution was dialyzed in ultrapure water for 3 days (3 times / day, 2 L / time) by using a dialysis bag with a molecular weight cut-off of 8000-14000, and then freeze-dried to obtain PEI.NH2-mPEG. The molar ratio of PEI.NH2, mPEG-COOH, EDC and NHS was 1:20:200:200.

[0056] (2) Step 2, 73 mg PEI.NH2-mPEG was dissolved in 10 mL DMSO, 2.8 mg FI was dissolved in 3 mL DMSO. The FI solution was added dropwise to the PEI.NH2-mPEG solution and stirred for 24 h in the dark. After the reaction was completed, the above solution was dialyzed in ultrapure water for 3 days (3 times / day, 2 L / time) by using a dialysis bag with a molecular weight cut-off of 8000-14000, and then freeze-dried to obtain PEI.NH2-mPEG-FI. The molar ratio of PEI.NH2-mPEG and FI was 1:5.

[0057] (3) Step 3, 32 mg PEI.NH2-mPEG-FI was dissolved in 5 mL ultrapure water, 5.2 mg CuCl2·2H2O was dissolved in 5 mL ultrapure water, 22.1 mg Na2S·9H2O was dissolved in 5 mL ultrapure water. The CuCl2 solution was added dropwise to the PEI.NH2-mPEG-FI solution and stirred for 15 min in the dark, then the Na2S solution was added dropwise to the above solution and stirred for 30 min in the dark at 70°C to form a dark green solution. After the reaction was completed, 64.4 mL N(C2H5)3 was added and stirred for 30 min in the dark, then 42.5 mL Ac2O was added and stirred for 24 h in the dark. After the reaction was completed, the above solution was dialyzed in ultrapure water for 3 days (3 times / day, 2 L / time) by using a dialysis bag with a molecular weight cut-off of 8000-14000, and then freeze-dried to obtain CuS PENs. The molar ratio of PEI.NH2-mPEG-FI, CuCl2·2H2O and Na2S·9H2O was 1:50:150, and the molar ratio of PEI.NH2-mPEG-FI, N(C2H5)3 and Ac2O was 1:500:500.

[0058] (4) Step 4, 12.5 mg CuS PENs were weighed and dissolved in 5 mL ultrapure water, then 280 mL of 8 mM FeCl3 solution was added to the CuS PENs solution, vortexed and mixed, then 557 mL of 8 mM EGCG solution was added, stirred in the dark for 24 h, after the reaction was completed, the above solution was subjected to ultrafiltration centrifugation (8000 rpm, 10 min) using an ultrafiltration centrifuge tube with a molecular weight cut-off of 3000, and the product solution obtained after ultrafiltration purification was freeze-dried to obtain CuS PENs@Fe-EGCG nanocomposites. The molar ratio of CuS PENs, FeCl3 and EGCG was 1:10:20.

[0059] The intermediate product of the comparative nanomaterial was used 1 H NMR was used for characterization, and the characteristic peaks in each 1 H NMR spectrum were integrated and calculated to know that each molecule of PEI.NH2-mPEG-Fl was modified with 13.3 mPEG-COOH and 2.3 Fl, see the accompanying drawings Figure 2 . Then UV-Vis spectrum scanning was performed, see the accompanying drawings Figure 3 , the ultraviolet-visible characteristic absorption peak of Fl was found at 500 nm, indicating that Fl was successfully modified on PEI.NH2-mPEG-Fl Figure 3 a) After CuSNPs were wrapped in PEI.NH2-mPEG-Fl and acetylated, the product CuS PENs was formed, and UV-Vis characterization was performed, and an absorption peak was found at 1000-1100 nm, which can be attributed to the characteristic peak of CuS NPs, thereby proving that CuS NPs were successfully wrapped in functionalized polyethyleneimine Figure 3 b).

[0060] Comparative Example 2

[0061] 15 mg of FA-CuS PENs prepared in Example 1 was weighed and dissolved in 5 mL ultrapure water, then 612 mL of 8 mM EGCG solution was added to the above solution, stirred in the dark for 24 h, after the reaction was completed, the above solution was subjected to ultrafiltration centrifugation (8000 rpm, 10 min) using an ultrafiltration centrifuge tube with a molecular weight cut-off of 3000, and the product solution obtained after ultrafiltration purification was freeze-dried to obtain FA-CuS PENs@EGCG nanocomposites. The molar ratio of FA-CuS PENs and EGCG was 1:20.

[0062] The following are the test results of the above-mentioned nanocomposites.

[0063] 1. Size and morphology characterization of the FA-CuS PENs@Fe-EGCG obtained in Example 1 and the FA-CuS PENs@EGCG obtained in Comparative Example 2. The FA-CuS PENs@Fe-EGCG and the FA-CuS PENs@EGCG were dissolved in ultrapure water to prepare a sample aqueous solution with a concentration of 1 mg / mL, and the TEM test results are shown in the accompanying drawings. Figure 4 It can be observed from the TEM and high-resolution TEM that the FA-CuS PENs@Fe-EGCG has a spherical structure, a uniform size distribution, and an average diameter of 10.81 ± 2.57 nm Figure 4 a, b, e). The TEM image of the FA-CuS PENs@EGCG shows that it also has a spherical structure, with an average diameter of 11.42 ± 2.49 nm Figure 4 c, f). After the FA-CuS PENs@Fe-EGCG was dissolved in a buffer solution with a pH of 6.5, TEM testing found that the FA-CuS PENs@Fe-EGCG particles had blurred boundaries and irregular shapes, which was due to the fact that the Fe-EGCG network surrounding the FA-CuS PENs had cracked under acidic conditions, causing changes in its morphology Figure 4 d).

[0064] In addition, the FA-CuS PENs@Fe-EGCG obtained in Example 1 was subjected to XPS testing to analyze the valence states of copper and iron, and the results are shown in the accompanying drawings. Figure 5 The XPS spectrum of the FA-CuS PENs@Fe-EGCG contains characteristic photoelectron lines of C1s, N 1s, O 1s, Fe 2p, and Cu 2p, indicating the presence of iron and copper elements in the FA-CuS PENs@Fe-EGCG, which means that the FA-CuS PENs@Fe-EGCG contains CuS NPs and a Fe-EGCG network Figure 5 a). After fine scanning of the FA-CuS PENs@Fe-EGCG, Cu 2p 3 / 2 peaks were found at 931.5 eV and 932.6 eV, Cu 2p 1 / 2 peaks were found at 951.2 eV and 952.4 eV, and a satellite peak of Cu(II) appeared at 942.6 eV, where the characteristic peaks at 931.5 eV and 951.2 eV belong to Cu(I), and the characteristic peaks at 932.6 eV and 952.4 eV belong to Cu(II). Fe 2p 3 / 2 peaks were found at 710.5 eV and 713.6 eV, and Fe 2p 1 / 2The satellite peaks of Fe(II) and Fe(III) appeared at 719.9 eV and 732.9 eV, respectively, and the characteristic peaks of Fe(II) were at 710.5 eV and 723.4 eV, and the characteristic peaks of Fe(III) were at 713.6 eV and 726.9 eV. The above results showed that Cu(I), Cu(II), Fe(II), and Fe(III) existed in the FA-CuS PENs@Fe-EGCG. Figure 5 b, c) The CuS NPs formed by Cu(I) and Cu(II) had photothermal effects and could be used for tumor PTT; Fe(II) existed because EGCG in the FA-CuS PENs@Fe-EGCG partially reduced Fe(III) to Fe(II), and the existence of Fe(II) and Fe(III) could induce tumor CDT.

[0065] 2, The photothermal performance of the FA-CuS PENs@Fe-EGCG obtained in Example 1 was evaluated. First, 100 μL of FA-CuS PENs@Fe-EGCG aqueous solution with different Cu concentrations (150 μM, 300 μM, 600 μM, and 1200 μM) was placed in a 500 μL EP tube, and ultrapure water was used as a control group. The solution in the EP tube was continuously irradiated with 1064 nm laser (1.0 W / cm 2 ) for 5 min, and the temperature change was monitored by a thermocouple (5 s / time). It was found that the temperature of the FA-CuS PENs@Fe-EGCG aqueous solution gradually increased with the increase of Cu concentration and the extension of laser irradiation time, and the results are shown in FIG. 2A of the specification. Figure 6 a. The temperature difference (ΔT) of the FA-CuS PENs@Fe-EGCG aqueous solution with different Cu concentrations before and after near-infrared laser (1064 nm, 1.0 W / cm 2 ) irradiation showed that the higher the Cu concentration, the greater the temperature difference, and the results are shown in FIG. 2B of the specification. Figure 6 b. Next, the FA-CuS PENs@Fe-EGCG aqueous solution concentration ([Cu]=600 μM, 100 μL) was selected, and the FA-CuS PENs@Fe-EGCG aqueous solution was irradiated with 1064 nm laser with different powers (0.6 W / cm 2 , 1.0 W / cm 2 , 1.5 W / cm 2 , and 2.0 W / cm 2 ) for 5 min, and the temperature change was monitored by a thermocouple (5 s / time). It was found that the temperature of the FA-CuS PENs@Fe-EGCG aqueous solution gradually increased with the increase of laser power density, and the results are shown in FIG. 2C of the specification. Figure 6 c.

[0066] Finally, the concentration of FA-CuS PENs@Fe-EGCG ([Cu]= 600 μM, 100 μL) and the laser power (1.0 W / cm 2 ) were selected, and the aqueous solution of FA-CuS PENs@Fe-EGCG was irradiated by 1064 nm laser for 250 s, then the laser was turned off and naturally cooled to room temperature. The temperature change was detected by thermocouple during the heating / cooling process (5 s / time). The heating / cooling process was repeated for 5 times. It was found that the highest temperature of each cycle of FA-CuS PENs@Fe-EGCG in the five laser cycles was about 55°C, and the highest temperature of the five cycles did not change significantly, indicating that FA-CuS PENs@Fe-EGCG had good photothermal stability Figure 6 d). Then the relationship curve between the natural cooling time of one cycle and the inverse of the natural logarithm of the temperature was calculated, which showed that the photothermal conversion efficiency of the aqueous solution of FA-CuS PENs@Fe-EGCG was 51.1%, and it had good photothermal conversion effect. The results are shown in Figs. 16e, f of the specification. Figure 6 e, f.

[0067] 3. In order to explore the in vitro release performance of Fe(III) and chemotherapeutic drug EGCG in FA-CuS PENs@Fe-EGCG, the release amount of Fe(III) and EGCG in FA-CuS PENs@Fe-EGCG under different pH conditions (pH = 6.5, pH = 7.4) and laser irradiation was analyzed by simulating the environment of normal tissues and tumor tissues in vivo. FA-CuS PENs@Fe-EGCG was dissolved in buffer solutions with different pH conditions (pH = 6.5, pH = 7.4) to obtain 3 mg / mL solutions with corresponding pH. The experiment was carried out under the conditions of pH = 7.4, pH = 6.5 and pH = 6.5 + laser, with three parallel samples in each group. Then 1 mL of the solution was taken and fixed in a dialysis bag with a molecular weight cutoff of 1000, and placed in a 50 mL centrifuge tube containing 19 mL of buffer solution with corresponding pH, and placed in a 37°C constant temperature incubator for shaking. For the laser group solution, the laser (1064 nm, 1.0 W / cm 2) and sampling. 1 mL of the outer solution was taken at different time points (0.5, 1, 2, 4, 8, 12, 24 and 48 h) for all groups, and 1 mL of fresh buffer of corresponding pH was added immediately to keep the total volume of the outer solution unchanged. The 1 mL of the outer solution taken at each time point was digested with aqua regia for 1 day and diluted with ultrapure water to 6 mL. The content of Fe element in the taken solution was measured by ICP-OES, and the release amount of Fe(III) was calculated by triplicate measurements. For the determination of the release amount of EGCG, the taken outer solution was measured by UV-Vis spectrophotometer for its absorbance value at 273 nm to calculate the release amount of EGCG through the standard curve of EGCG under different pH conditions. The test results are as follows Figure 7 a, b, within the first 12 h, Fe(III) and EGCG were released rapidly, and then the release of Fe(III) and EGCG gradually slowed down with the extension of time. At 48 h, the cumulative release amounts of Fe(III) and EGCG under the condition of pH = 6.5 reached 33.1% and 50.7%, respectively, and the release amounts of Fe(III) and EGCG under the condition of pH = 6.5 were significantly higher than those of Fe(III) and EGCG under the condition of pH = 7.4 (p < 0.001), because under the condition of low pH, the phenolic hydroxyl groups on the polyphenol were mostly protonated, and the competitive chelation was formed between the proton hydrogen and the metal ion and the phenolic hydroxyl groups on the polyphenol. Due to the occupation of the chelation of the proton hydrogen, the Fe-EGCG was unstable under the condition of low pH and dissociated, thereby promoting the release of Fe(III) and EGCG. In addition, under the condition of pH = 6.5, the FA-CuS PENs@Fe-EGCG solution was irradiated by laser (1064 nm, 1.0 W / cm 2 ) for 5 min, it can be found that the release amounts of Fe(III) and EGCG in the FA-CuS PENs@Fe-EGCG solution treated by laser were significantly higher than those in the FA-CuS PENs@Fe-EGCG solution without laser treatment under the condition of pH = 6.5 after 48 h (p < 0.01), which may be due to the temperature rise caused by laser promoting the self-dissociation of Fe-EGCG network, so that the release amounts of Fe(III) and EGCG increased.

[0068] 4, the iron component of the FA-CuS PENs@Fe-EGCG in Example 1 generates ROS by Fenton reaction in the environment of high endogenous hydrogen peroxide of cancer cells, and the environment is simulated in vitro and verified. Since ROS can cause the degradation of MB to cause the weakening of the ultraviolet-visible absorption peak of MB at 665 nm, the MB degradation experiment is often used to detect whether ROS is generated.

[0069] The experiment was first divided into the following groups: MB group, FA-CuS PENs@Fe-EGCG + H2O2+ MB group, FA-CuS PENs@Fe-EGCG + H2O2+ MB + GSH group, and FA-CuS PENs@Fe-EGCG + H2O2+ MB + laser (1064 nm, 1.0 W / cm 2 ) group. The specific operation process was as follows: H2O2 solution and MB solution were added to the FA-CuS PENs@Fe-EGCG aqueous solution to make the final concentration of Fe in the FA-CuS PENs@Fe-EGCG 6 μg / mL, the final concentration of H2O2 10 mM, and the final concentration of MB 10 μg / mL, and then the FA-CuS PENs@Fe-EGCG + H2O2+ MB mixed solution was stirred at room temperature in the dark for 6 h, which was the FA-CuS PENs@Fe-EGCG + H2O2+ MB group. At the same time, GSH solution was first added to the FA-CuS PENs@Fe-EGCG aqueous solution, and then H2O2 solution and MB solution were added to make the final concentration of Fe in the FA-CuS PENs@Fe-EGCG 6 μg / mL, the final concentration of GSH 10 mM, the final concentration of H2O2 10 mM, and the final concentration of MB 10 μg / mL, and finally the FA-CuS PENs@Fe-EGCG + H2O2+ MB + GSH mixed solution was stirred at room temperature in the dark for 6 h, which was the FA-CuS PENs@Fe-EGCG + H2O2+ MB + GSH group. For the laser treatment group solution, H2O2 solution and MB solution were first added to the FA-CuS PENs@Fe-EGCG aqueous solution to make the final concentration of Fe in the FA-CuS PENs@Fe-EGCG 6 μg / mL, the final concentration of H2O2 10 mM, and the final concentration of MB 10 μg / mL, and then the above mixed solution was irradiated with laser (1064 nm, 1.0 W / cm 2 ) for 5 min, and finally the FA-CuS PENs@Fe-EGCG + H2O2+ MB + laser group mixed solution was stirred at room temperature in the dark for 6 h, which was the FA-CuS PENs@Fe-EGCG + H2O2+ MB + laser group. At the same time, pure MB solution was used as a control, and the final concentration of MB was 10 μg / mL. The above four groups of reaction solutions were scanned by UV-Vis spectroscopy, respectively. The results are shown in the accompanying drawings Figure 8As shown in Fig. a, the absorbance of FA-CuS PENs@Fe-EGCG+H2O2+MB mixed solution at 665 nm slightly decreased compared with that of pure MB solution at 665 nm, indicating that Fe(III) in FA-CuS PENs@Fe-EGCG could generate ROS in the presence of H2O2, thereby leading to the degradation of MB. In addition, the absorbance of FA-CuS PENs@Fe-EGCG+H2O2+MB+GSH mixed solution at 665 nm significantly decreased compared with that of FA-CuS PENs@Fe-EGCG+H2O2+MB mixed solution at 665 nm, because GSH could react with Fe(III) in FA-CuS PENs@Fe-EGCG to generate Fe(II), and the generated Fe(II) could react with H2O2 to generate ·OH, thereby leading to more degradation of MB. The absorbance of FA-CuS PENs@Fe-EGCG+H2O2+MB+laser mixed solution at 665 nm significantly decreased compared with that of FA-CuS PENs@Fe-EGCG+H2O2+MB mixed solution at 665 nm, because the temperature rise caused by CuS NPs-mediated PTT in FA-CuS PENs@Fe-EGCG under laser irradiation could accelerate the generation of Fe(III)-induced ROS, ultimately leading to more degradation of MB.

[0070] Next, the FA-CuS PENs@Fe-EGCG aqueous solution was mixed with H2O2 solution and MB solution, so that the final concentration of Fe in FA-CuS PENs@Fe-EGCG was 6 μg / mL, the final concentration of H2O2 was 10 mM, and the final concentration of MB was 10 μg / mL. Then, UV-Vis spectrum scanning was performed at different reaction time points (6 h, 12 h, 18 h, 24 h). The degradation curve of MB with time change was found as shown in Fig. b, indicating that the generated ROS also gradually increased with time, thereby leading to an increase in the degree of MB degradation. Figure 8 b shows that the generated ROS also gradually increased with time, thereby leading to an increase in the degree of MB degradation.

[0071] Moreover, the degradation effect of the mixed solution of FA-CuS PENs@Fe-EGCG and H2O2 on MB under different pH (6.5, 7.4) conditions was studied. The H2O2 and MB solutions corresponding to the pH buffer were added into the FA-CuS PENs@Fe-EGCG solution dissolved in different pH buffers (6.5, 7.4), so that the final concentration of Fe in the FA-CuS PENs@Fe-EGCG was 6 μg / mL, the final concentration of H2O2 was 10 mM, and the final concentration of MB was 10 μg / mL under different pH (6.5, 7.4) conditions. Then, the absorbance of each group of solutions at 665 nm was tested by UV-Vis spectroscopy at different reaction time points (0 h, 1 h, 2 h, 3 h, 4 h), and the residual rate of MB was calculated. It was found that the residual rate of MB decreased significantly with the decrease of pH, and the residual rate of MB in the FA-CuS PENs@Fe-EGCG+H2O2+MB solution under pH = 6.5 was significantly lower than that under pH = 7.4 (p < 0.01) after 4 h of MB degradation, indicating that the release of Fe(III) in FA-CuS PENs@Fe-EGCG increased in an acidic environment, which was conducive to the generation of more ROS by FA-CuS PENs@Fe-EGCG, resulting in more MB degradation. Figure 8 c).

[0072] Finally, the role of EGCG in the Fenton reaction was explored. EGCG solution, H2O2 solution and MB solution were added to the FeCl3 solution, so that the final concentration of Fe in FeCl3 was 6 μg / mL, the final concentration of EGCG was 24 μg / mL, the final concentration of H2O2 was 10 mM, and the final concentration of MB was 10 μg / mL. Then, the UV-Vis spectrum was tested after incubation at room temperature for 12 h. Meanwhile, H2O2 solution and MB solution were added to the FeCl3 solution, so that the final concentration of Fe in FeCl3 was 6 μg / mL, the final concentration of H2O2 was 10 mM, and the final concentration of MB was 10 μg / mL, and the FeCl3+H2O2+MB mixed solution was used as a control. As shown in FIG. 16D, the absorbance of the FeCl3+EGCG+H2O2+MB mixed solution at 665 nm was lower than that of the FeCl3+H2O2+MB mixed solution, indicating that EGCG promoted the conversion of Fe(III) to Fe(II), accelerated the generation of ROS, and led to an increase in the degree of MB degradation. Figure 8 d.

[0073] 5、To detect the MR imaging performance of FA-CuS PENs@Fe-EGCG, aqueous solutions of FA-CuS PENs@Fe-EGCG with Fe concentrations of 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM and 0.8 mM were prepared, and then MR imaging was performed on them and the relaxation time (T1) of each Fe concentration solution was obtained. The reciprocal of the measured relaxation time (1 / T1) was fitted with the Fe concentration of FA-CuS PENs@Fe-EGCG, and the slope of the obtained fitting curve was the relaxation rate (r1) of FA-CuS PENs@Fe-EGCG. The results are shown in FIGS. 1A and 1B of the accompanying drawings. Figure 9 a, as the Fe concentration increases, the signal intensity of the MR imaging pseudo-color map gradually increases. Linear fitting of the Fe concentration and 1 / T1 shows that the T1 relaxation rate r1 of FA-CuS PENs@Fe-EGCG is 4.8005 mM-1s-1, and the R2 value of the fitting curve is 0.9999, as shown in FIGS. 2A and 2B of the accompanying drawings. Figure 9 b. -1 s -1 .

[0074] 6、FA-CuS PENs@Fe-EGCG, FA-CuS PENs@EGCG and CuS PENs@Fe-EGCG were dispersed in H2O respectively to obtain aqueous solutions with a concentration of 0.05 mg / mL. Dynamic light scattering was used to characterize the hydration particle size of FA-CuS PENs@Fe-EGCG, FA-CuS PENs@EGCG and CuS PENs@Fe-EGCG aqueous solutions at 1, 3, 5 and 7 days respectively. It was found that the hydration particle sizes of FA-CuS PENs@Fe-EGCG, FA-CuS PENs@EGCG and CuS PENs@Fe-EGCG at different time points (1, 3, 5 and 7 days) did not change significantly, indicating that FA-CuS PENs@Fe-EGCG, FA-CuS PENs@EGCG and CuS PENs@Fe-EGCG have certain colloidal stability, as shown in FIG. 3A of the accompanying drawings. Figure 10 a. Subsequently, the photothermal stability of FA-CuS PENs@Fe-EGCG was characterized by UV-Vis testing. An aqueous solution of FA-CuS PENs@Fe-EGCG with a concentration of 0.4 mg / mL was irradiated with near-infrared laser (1064 nm, 1 W / cm 2 ) for 5 min, and an aqueous solution of FA-CuS PENs@Fe-EGCG (0.4 mg / mL) without laser irradiation was used as a control. UV-Vis testing found that the UV-Vis spectra of the aqueous solution of FA-CuS PENs@Fe-EGCG before and after laser irradiation did not change, indicating that FA-CuS PENs@Fe-EGCG has good photothermal stability, as shown in FIG. 3B of the accompanying drawings. Figure 10 b.

[0075] 7、Good hemocompatibility is the basis for subsequent animal experiments, so the hemolysis of FA-CuS PENs@Fe-EGCG on red blood cells was studied. The blood of healthy nude mice was collected, 1 mL of blood was collected by centrifugation (3000 rpm, 5 min), and then resuspended with PBS buffer and centrifuged, and the resuspension and centrifugation operation was repeated until the cell supernatant was clear, and then the obtained cell pellet was resuspended with 30 times the volume of PBS buffer to form a red blood cell suspension. Take 7 1.5 mL centrifuge tubes, add 500 μL of red blood cell suspension to each tube and add 500 μL of ultrapure water, PBS buffer and different Fe concentrations (5, 10, 25, 50, 100 μM) FA-CuS PENs@Fe-EGCG PBS buffer respectively, mix uniformly and incubate at 37°C for 2h. After incubation, centrifuge (10000 rpm, 5 min), take pictures of each sample and measure the absorbance of each group of sample supernatant at 540 nm to calculate the hemolysis rate. The red blood cells treated with PBS buffer are negative control, and the red blood cells treated with ultrapure water are positive control. The hemolysis rate is calculated by the following formula:

[0076] Hemolysis rate (%) = (Ds-Db-Dnc) / (Dpc-Dnc) x 100%,

[0077] Figure 11 The results are shown in the accompanying drawings. The red blood cells treated with ultrapure water are positive control, and the red blood cells treated with PBS are negative control. It is found that in the Fe concentration range of 5-100 μM, with the increase of Fe concentration, the hemolysis rate of red blood cells treated with FA-CuS PENs@Fe-EGCG at each Fe concentration is below 5%. At the same time, the red blood cells treated with ultrapure water in the insert have obvious cell rupture, and the red blood cells treated with PBS and FA-CuS PENs@Fe-EGCG ([Fe] = 5-100 μM) do not appear cell rupture, indicating that FA-CuS PENs@Fe-EGCG has good hemocompatibility.

[0078] 8、To explore the targeting specificity of FA-CuS PENs@Fe-EGCG for cancer cells with high expression of folate receptor, mouse breast cancer 4T1 cells with folate receptor expression were selected as model cells for flow cytometry test. First, 2 x 10 5 ​Cell density of cells per well 4T1 cells were seeded in 12-well plates and incubated for 24 h, then the original culture medium was removed and the cells were divided into two groups, one group was added with fresh culture medium containing FA (2.5 μM) and continued to incubate for 2 h to saturate the folate receptor. The other group was added with the same amount of fresh culture medium without FA and also continued to incubate for 2 h. After incubation, the culture medium was removed to obtain 4T1 cells with low folate receptor expression (4T1-LFAR) and 4T1 cells with high folate receptor expression (4T1-HFAR), respectively. Then 1 mL of FA-CuS PENs@Fe-EGCG, CuS PENs@Fe-EGCG with CuS concentration of 0.32 mM were added to incubate 4T1-HFAR cells and 4T1-LFAR cells, respectively, and 4T1-HFAR cells incubated with culture medium containing equal volume of PBS were used as a control, each group had three parallel samples. After 4 h of incubation, the culture medium was removed and washed with PBS for 2-3 times to remove residual culture medium. Then trypsin was added and placed in a cell incubator to digest the cells for 3-5 min, fresh culture medium was added to terminate the digestion and suspend the cells by blowing. The cells in suspension were transferred to a centrifuge tube, after centrifugation, the obtained cells were resuspended with 1 mL of PBS to obtain five groups of cell suspensions including 4T1-HFAR cells and 4T1-LFAR cells incubated with FA-CuS PENs@Fe-EGCG culture medium, 4T1-HFAR cells and 4T1-LFAR cells incubated with CuS PENs@Fe-EGCG culture medium, and 4T1-HFAR cells incubated with PBS culture medium. Finally, based on the fluorescence of FI in the nanomaterials, each group of cell samples was measured by flow cytometry in the FL1 fluorescence channel (488 nm), and 10 4 Figure 12 Figure 12 Figure 12 Figure 13 ​​​​a) Quantitative analysis of the uptake rate of 4T1 cells (percentage of cells that uptake nanocomposites in each sample of statistical cells) can find that the cell uptake rate of 4T1-HFAR cells treated with FA-CuSPENs@Fe-EGCG is significantly higher than that of 4T1-LFAR cells treated with FA-CuSPENs@Fe-EGCG (p<0.001), which is 8.5 times that of 4T1-LFAR cells treated with FA-CuSPENs@Fe-EGCG Figure 13 b) The above results show that due to the specific binding characteristics of FA and FA receptors, FA-CuSPENs@Fe-EGCG modified by FA can significantly increase the uptake of 4T1-HFAR cancer cells.

[0079] 9) The fluorescence of FI in FA-CuSPENs@Fe-EGCG can be directly observed by CLSM, which can also be used to evaluate the targeted specific uptake ability of FA-CuSPENs@Fe-EGCG by cancer cells with high expression of folate receptors. First, 1×10 5 The 4T1 cells were seeded in a 12-well plate with a coverslip at a cell density of 1×104cells / well and incubated for 24 h, then the original culture medium was removed and the cells were divided into two groups, 4T1-LFAR cells and 4T1-HFAR cells, according to the method in Example 9. 1 mL of FA-CuSPENs@Fe-EGCG, CuSPENs@Fe-EGCG with a CuS concentration of 0.32 mM was added to the culture medium of 4T1-HFAR cells and 4T1-LFAR cells, respectively, and 4T1-HFAR cells incubated with culture medium containing equal volume of PBS were used as a control, each group had three parallel samples. After 4 h of incubation, the culture medium was removed and the cells were washed 2-3 times with PBS to remove residual culture medium. Then 500 μL of 2.5% glutaraldehyde was added to each well at 4°C to fix the cells, and after 15 min the cells were washed 2-3 times with cold PBS to remove glutaraldehyde. Then 1 mL of DAPI staining agent with a concentration of 1 μg / mL was added to each well at room temperature to stain the cells in each group, and after 20 min the excess staining agent was washed with PBS. Finally, the coverslips containing the cell samples in each group were mounted on a glass slide, and the obtained cell samples were observed and photographed using a CLSM system. The test results are shown in the accompanying drawings Figure 14As shown, compared with PBS-treated 4T1-HFAR cells, 4T1-LFAR cells and 4T1-HFAR cells treated with CuS PENs@Fe-EGCG showed only weak green fluorescence, 4T1-LFAR cells treated with FA-CuS PENs@Fe-EGCG also showed weak green fluorescence, while 4T1-HFAR cells treated with FA-CuS PENs@Fe-EGCG showed extremely significant green fluorescence. The difference in fluorescence intensity of cells in each group further illustrates that due to the specific targeting effect of FA, 4T1-HFAR cells increase the uptake of FA-CuS PENs@Fe-EGCG.

[0080] 10. Since FA-CuS PENs@Fe-EGCG contains Fe element, the uptake of Fe element by 4T1 cells can be tested by ICP-OES to indirectly evaluate the targeted specific uptake ability of FA-CuS PENs@Fe-EGCG by cancer cells with high expression of folate receptor. First, 2 x 10 5Cell density of cells per well 4T1-HFAR cells and 4T1-LFAR cells were seeded in 12-well plates, FA-CuS PENs@Fe-EGCG was dissolved in culture medium and diluted step by step to obtain fresh culture medium containing different Fe concentrations (0 μM, 10 μM, 20 μM, 40 μM, 80 μM, 100 μM) of FA-CuS PENs@Fe-EGCG. Then 4T1-HFAR cells and 4T1-LFAR cells were incubated with the above-mentioned fresh culture medium containing materials, respectively, and each group had three parallel samples. After 4 h of incubation, the culture medium was removed and washed with PBS for 2-3 times to remove residual culture medium. Then trypsin was added to each well of the cell culture plate and the plate was placed in the cell culture incubator to digest the cells. After 3-5 min, the cell culture plate was taken out, fresh culture medium was added to each well to stop the digestion and the suspended cells were blown. Then the cells in suspension were transferred to a centrifuge tube and centrifuged at a speed of 900-1000 rpm for 3 min, the supernatant was discarded and the cells were resuspended with 1 mL of PBS solution. Then 2 mL of aqua regia was added to the obtained cell suspension to lyse the cells, and after 24 h, the obtained cell lysate was diluted to 6 mL using PBS. Finally, the Fe content in each group of cell lysate dilutions was detected using ICP-OES, and the average Fe uptake of each group of cells was further calculated. The results showed that as the Fe concentration in FA-CuS PENs@Fe-EGCG increased, the Fe content in 4T1-LFAR cells and 4T1-HFAR cells treated with FA-CuS PENs@Fe-EGCG gradually increased, and when [Fe]≥40 μM, the Fe content in 4T1-HFAR cells treated with FA-CuS PENs@Fe-EGCG was significantly higher than that in 4T1-LFAR cells treated with FA-CuS PENs@Fe-EGCG (p<0.05, Figure 15 ), which indicated that FA-CuS PENs@Fe-EGCG could specifically bind to cancer cells with high expression of folate receptor and enhance the uptake of 4T1-HFAR cancer cells to the material.

[0081] 11、Based on the paramagnetic property of iron ions, MR imaging can be used, and FA-CuS PENs@Fe-EGCG contains Fe(III) and FA, so it is necessary to explore its MR imaging performance for targeting cancer cells. 2×10 6Cell density of cells per well 4T1 cells were seeded in 6-well plates for 24 h. Before the end of cell incubation, cell culture medium containing different Fe concentrations (0, 0.1, 0.2, 0.4, 0.8 mM) of FA-CuS PENs@Fe-EGCG and CuS PENs@Fe-EGCG was prepared. The original culture medium was removed and the above newly prepared culture medium containing different iron ion concentrations was added. After 4 h of incubation, the culture medium was removed, and three parallel samples were prepared for each group, and then washed with PBS for 2-3 times to remove the residual culture medium, to obtain 4T1 cells incubated with different Fe concentrations of FA-CuS PENs@Fe-EGCG and CuS PENs@Fe-EGCG, respectively. Trypsin was added to each group of cell samples, and the plate was placed in a cell incubator for digestion. After 3-5 min, it was taken out of the incubator and fresh medium was added to terminate the digestion, and then the suspended cells were blown. Subsequently, the cells in the suspended state were transferred to a centrifuge tube and centrifuged at a speed of 900-1000 rpm for 3 min. The supernatant was discarded and the cells were resuspended with 100 μL PBS. Finally, the collected 4T1 cell suspension was subjected to in vitro MR imaging using the MR imaging system. The test results are shown in the accompanying drawings Figure 16 As shown in the accompanying drawings, with the increase of Fe concentration in CuS PENs@Fe-EGCG and FA-CuS PENs@Fe-EGCG, the MR imaging signal intensity of the 4T1 cells incubated with them also gradually increased Figure 16 a), and then quantitative analysis of the MR imaging signal intensity showed that the MR signal value gradually increased with the increase of Fe concentration. When the Fe concentration was 0.4 mM and 0.8 mM, the MR signal value of the 4T1 cells treated with FA-CuS PENs@Fe-EGCG was 1.17 and 1.23 times that of the 4T1 cells treated with CuS PENs@Fe-EGCG under the same Fe concentration Figure 16 b). The above results prove that the modification of FA improves the T1-weighted MR imaging effect of 4T1 cells incubated with FA-CuS PENs@Fe-EGCG, which is caused by the FA-mediated targeting of cancer cell receptor-ligand endocytosis, and can achieve in vitro targeted cancer cell MR imaging.

[0082] 12. In order to explore the cytotoxicity of the prepared nanomaterials on normal mouse fibroblast (L929) cells and 4T1 cancer cells, CCK-8 test was performed. First, the cytotoxicity of the prepared nanomaterials on L929 cells was investigated. The L929 cells were seeded in 96-well plates at a density of 1.0 × 10 4Cells were seeded at a density of 10% per well in 96-well cell culture plates and incubated overnight at 37°C with 5% CO2. After discarding the culture medium, each well was replaced with 100 μL of MEM medium containing different CuS concentrations (0, 0.08, 0.16, 0.24, 0.32, 0.4, 0.48 mM) of FA-CuS PENs@EGCG, CuS PENs@Fe-EGCG, and FA-CuS PENs@Fe-EGCG, with three replicates for each concentration, and incubated for 24 h. Subsequently, the original culture medium was discarded, and 100 μL of serum-free medium containing 10% CCK-8 was added to each well. After culturing for another 4 h, the cell culture plates were placed in a multi-plate microplate reader, and the absorbance was measured at 450 nm to calculate the cell viability of L929 cells. The test results showed that as the CuS concentration increased, the cell viability of L929 cells treated with FA-CuS PENs@Fe-EGCG, CuSPENs@Fe-EGCG, and FA-CuS PENs@EGCG were all above 85.2%, indicating that FA-CuS PENs@Fe-EGCG, CuS PENs@Fe-EGCG, and FA-CuS PENs@EGCG had no significant inhibitory effect on normal cells. Figure 17 a).

[0083] Next, the cytotoxicity of the different prepared nanomaterials on 4T1 cells was investigated, according to a concentration of 1.0 × 10⁻⁶. 4 Cell density per well: 4T1 cells were seeded in 96-well cell culture plates and incubated overnight at 37°C with 5% CO2. After discarding the culture medium, each well was replaced with 100 μL of DMEM medium containing different CuS concentrations (0, 0.08, 0.16, 0.24, 0.32, 0.4, 0.48 mM) of FA-CuS PENs@Fe-EGCG, CuS PENs@Fe-EGCG, FA-CuS PENs@EGCG, FA-CuS PENs, and CuS PENs, with three replicates per concentration, and incubated for 24 h. The original culture medium was then discarded, and 100 μL of serum-free medium containing 10% CCK-8 was added to each well. After culturing for another 4 h, the cell culture plates were placed in a multi-plate reader, and the absorbance was measured at 450 nm to calculate the cell viability of the 4T1 cells. Results are as follows: Figure 17As shown in FIG. 8B, the cell viability of 4T1 cells treated with CuS PENs and FA-CuS PENs was higher than 83.2% in the range of 0-0.48 mM CuS concentration, indicating that CuS PENs and FA-CuS PENs had no 4T1 cell inhibition effect without laser irradiation. When [CuS]≥0.08 mM, the cell viability of 4T1 cells treated with FA-CuS PENs@EGCG was significantly lower than that of 4T1 cells treated with FA-CuS PENs (p<0.001) at the same CuS concentration, indicating that the EGCG-mediated chemotherapy in FA-CuS PENs@EGCG had a certain inhibition effect on cancer cells. When [CuS]≥0.08 mM, the cell viability of 4T1 cells treated with FA-CuS PENs@Fe-EGCG was significantly lower than that of 4T1 cells treated with FA-CuS PENs@EGCG (p<0.01) at the same CuS concentration, indicating that the combined treatment of cancer cells caused by EGCG-mediated chemotherapy and Fe(III)-mediated CDT in FA-CuS PENs@Fe-EGCG was obviously better than the single chemotherapy effect of EGCG-mediated chemotherapy in FA-CuS PENs@EGCG. When [CuS]≥0.32 mM, the cell viability of 4T1 cells treated with FA-CuS PENs@Fe-EGCG was significantly lower than that of 4T1 cells treated with CuS PENs@Fe-EGCG (p<0.05) at the same CuS concentration, indicating that due to the targeting effect of FA, the uptake of 4T1 cells to FA-CuS PENs@Fe-EGCG was increased, thereby enhancing the cancer cell inhibition effect of FA-CuS PENs@Fe-EGCG.

[0084] Next, the inhibition effect of each CuS NPs-containing nanomaterial-mediated photothermal therapy on 4T1 cells was explored. 1.0×10 4The density of cells per well was 4T1 cells were seeded in 96-well plates and cultured overnight, and then the culture medium was discarded. Subsequently, the 4T1 cells were divided into the following groups: PBS group, PBS+laser group, FA-CuS PENs@Fe-EGCG group, FA-CuS PENs@Fe-EGCG+laser group, CuS PENs@Fe-EGCG group, CuS PENs@Fe-EGCG+laser group, FA-CuS PENs@EGCG group, FA-CuS PENs@EGCG+laser group, FA-CuS PENs group, and FA-CuS PENs+laser group. That is, 4T1 cells were incubated with fresh culture medium containing FA-CuS PENs@Fe-EGCG, CuS PENs@Fe-EGCG, FA-CuS PENs@EGCG, and FA-CuS PENs at a CuS concentration of 0.32 mM for 24 h, and 4T1 cells incubated with PBS for 24 h were used as controls. Three parallel samples were set for each group. After incubation, the cells in each group were washed with PBS and fresh DMEM medium was added. For 4T1 cells in the laser irradiation group (+laser), the cells were irradiated with near-infrared laser (1064 nm, 1.0 W / cm 2 ) for 300 s after incubation with the materials. After the above cells were treated differently, the culture medium in each well of each experimental group was replaced with 100 μL of serum-free culture medium containing 10% CCK-8, and incubation was continued at 37°C in a 5% CO2 environment for 4 h. Finally, the absorbance of each well at 450 nm was measured using a microplate reader, and the cell viability of 4T1 cells was calculated. The test results are shown in Figure 17 c. The cell viability of 4T1 cells treated with each group of nanomaterials (FA-CuS PENs@Fe-EGCG, CuS PENs@Fe-EGCG, FA-CuS PENs@EGCG, and FA-CuS PENs) and irradiated with laser (1064 nm, 1.0 W / cm 2 ) for 5 min was significantly lower than that of 4T1 cells treated with the corresponding nanomaterials without laser irradiation (p<0.001), indicating that the photothermal effect of CuS NPs contained in each group of materials under laser irradiation can greatly inhibit cancer cells and has a significant photothermal treatment effect. Finally, in order to evaluate the treatment efficiency of cancer cells in each treatment group, the cell viability of 4T1 cells treated with each group of nanomaterials under laser (+laser) irradiation was compared, and the results are shown in Figure 17It can be seen that the 4T1 cell viability of FA-CuS PENs@EGCG + laser treatment was significantly lower than that of FA-CuS PENs + laser treatment (p < 0.001), indicating that under laser irradiation, the combined treatment effect of EGCG-mediated chemotherapy and CuS NPs-mediated PTT in FA-CuS PENs@EGCG was stronger than the PTT effect of CuS NPs in FA-CuS PENs. The 4T1 cell viability of FA-CuS PENs@Fe-EGCG + laser treatment was significantly lower than that of FA-CuS PENs@EGCG + laser treatment (p < 0.001), indicating that under laser irradiation, the combined treatment effect of EGCG-mediated chemotherapy, CuS NPs-mediated PTT, and Fe(III)-mediated CDT in FA-CuS PENs@Fe-EGCG was stronger than the combined treatment effect of EGCG-mediated chemotherapy and CuS NPs-mediated PTT in FA-CuS PENs@EGCG. The 4T1 cell viability of FA-CuS PENs@Fe-EGCG + laser treatment was significantly lower than that of CuS PENs@Fe-EGCG + laser treatment (p < 0.001), indicating that even though both FA-CuS PENs@Fe-EGCG + laser and CuS PENs@Fe-EGCG + laser have the effect of combined chemotherapy / photothermal / chemical kinetics treatment, the targeting effect of FA in FA-CuS PENs@Fe-EGCG + laser increases the uptake of FA-CuS PENs@Fe-EGCG by 4T1 cells, resulting in more 4T1 cell death, and it has a stronger 4T1 cell inhibition effect.

[0085] 13、In order to further explore the apoptosis mechanism mediated by each material group (FA-CuS PENs@EGCG, FeCl3, CuS PENs@Fe-EGCG, FA-CuS PENs@Fe-EGCG, FA-CuS PENs@Fe-EGCG + laser), each group of nanomaterials without FI modification was used for Annexin V-FITC / PI double staining test of 4T1 cells. First, 2.0 x 10 5The 4T1 cells were seeded in 12-well plates at a density of 1 cell / well and incubated overnight, then the culture medium was discarded and the culture medium containing CuS at a concentration of 0.32 mM was added to the FA-CuS PENs@EGCG, CuS PENs@Fe-EGCG, FA-CuS PENs@Fe-EGCG and FeCl3 containing Fe at a concentration of 80 μM, respectively, and incubated for 24 h. The culture medium containing FeCl3 ([Fe]=80 μM) was equivalent to the Fe concentration in the culture medium containing CuS PENs@Fe-EGCG and FA-CuS PENs@Fe-EGCG at a CuS concentration of 0.32 mM. At the same time, 4T1 cells treated with PBS were used as a control. After incubation, the culture medium was discarded and the cells were washed with PBS for 2-3 times. The 4T1 cells incubated with FA-CuS PENs@Fe-EGCG were divided into two groups, i.e. the laser (1064 nm, 1.0 W / cm 2 , 5 min) irradiated cell group and the non-laser irradiated cell group. After the cells treated with each material were treated with / without laser irradiation, the cells in each group were collected by digestion and resuspended in 300 μL of pre-cooled buffer, then 5 μL of Annexin V-FITC and 5 μL of PI solution were added to each cell sample in turn according to the standard experimental operation in the kit instructions and incubated at room temperature in the dark for 15 min. Finally, the flow cytometry was used for testing. It can be seen from the double staining results of flow cytometry that compared with the apoptosis degree of 4T1 cells treated with PBS, the 4T1 cells in the FA-CuS PENs@EGCG, FeCl3, CuS PENs@Fe-EGCG, FA-CuS PENs@Fe-EGCG and FA-CuS PENs@Fe-EGCG+laser groups all appeared a certain degree of apoptosis, and the apoptosis degree of 4T1 cells in the FA-CuS PENs@Fe-EGCG+laser group was the largest Figure 18a) The percentage of apoptotic cells (early and late apoptotic cells) in each treatment group was statistically analyzed by flow cytometry. The percentage of apoptotic cells was ranked as follows: PBS group < FA-CuS PENs@EGCG group < FeCl3 group < CuS PENs@Fe-EGCG group < FA-CuS PENs@Fe-EGCG group < FA-CuS PENs@Fe-EGCG + laser group treated 4T1 cells. The percentage of apoptotic cells in 4T1 cells treated with FA-CuS PENs@Fe-EGCG was significantly higher than that in 4T1 cells treated with FA-CuS PENs@EGCG (p < 0.001), indicating that the degree of apoptosis caused by the combination of EGCG-mediated chemotherapy and Fe(III)-mediated CDT in FA-CuS PENs@Fe-EGCG was higher than that caused by EGCG-mediated chemotherapy in FA-CuS PENs@EGCG. The percentage of apoptotic cells in 4T1 cells treated with FA-CuS PENs@Fe-EGCG + laser was significantly higher than that in 4T1 cells treated with FA-CuS PENs@EGCG (p < 0.001), indicating that under laser irradiation, the degree of apoptosis caused by the combination of EGCG-mediated chemotherapy, CuS NPs-mediated PTT, and Fe(III)-mediated CDT in FA-CuS PENs@Fe-EGCG was higher than that caused by EGCG-mediated chemotherapy alone in FA-CuS PENs@EGCG. Meanwhile, the percentage of apoptotic cells in 4T1 cells treated with FA-CuS PENs@Fe-EGCG was significantly higher than that in 4T1 cells treated with CuS PENs@Fe-EGCG (p < 0.001), indicating that under the condition of both FA-CuS PENs@Fe-EGCG and CuS PENs@Fe-EGCG having chemotherapy / chemodynamic combination therapy, the active targeting effect of FA increased the uptake of 4T1 cells to FA-CuS PENs@Fe-EGCG. Therefore, compared with CuS PENs@Fe-EGCG, FA-CuS PENs@Fe-EGCG can induce more apoptosis of cancer cells.Finally, the percentage of apoptosis of 4T1 cells treated with FA-CuS PENs@Fe-EGCG + laser was 48.0%, which was significantly higher than that of 4T1 cells treated with FA-CuS PENs@Fe-EGCG (35.1%, p < 0.001), indicating that the degree of apoptosis caused by the combined treatment of EGCG-mediated chemotherapy, CuS NPs-mediated PTT and Fe(III)-mediated CDT in FA-CuS PENs@Fe-EGCG + laser was higher than that of the combined treatment of EGCG-mediated chemotherapy and Fe(III)-mediated CDT in FA-CuS PENs@Fe-EGCG. Figure 18 b). This result is consistent with the results of the CCK-8 test in Example 13, in which the viability of 4T1 cells treated with FA-CuS PENs@Fe-EGCG after laser irradiation was lower than that of 4T1 cells treated with FA-CuS PENs@Fe-EGCG without laser irradiation at a CuS concentration of 0.32 mM.

[0086] 14. To further explore the mechanism behind the induction of apoptosis in 4T1 cells by FA-CuS PENs@Fe-EGCG, the ROS content in 4T1 cells treated with different material groups (FA-CuS PENs@EGCG, FeCl3, CuS PENs@Fe-EGCG, FA-CuS PENs@Fe-EGCG, FA-CuS PENs@Fe-EGCG + laser) was explored using the DCFH-DA fluorescent probe. Similarly, 4T1 cells were incubated with each group of unmodified FI to avoid interference with the experimental results by the fluorescence of FI itself. First, 4T1 cells were seeded in a 12-well plate at a density of 2.0 x 10 5 After 4T1 cells were seeded in a 12-well plate at a density of 2.0 x 10 2, 5 min) irradiated cell groups and non-laser irradiated cell groups. After the cells in each group were treated with / without laser irradiation, 1 mL of serum-free medium containing 10 μM DCFH-DA was added to each well, and the cells were incubated in the incubator for 40 min in the dark to obtain 6 groups of cell samples, with 3 parallel samples in each group. Then, the cells were washed, digested, collected in flow tubes, and tested using a flow cytometer. The results of the flow cytometry test showed that, compared with the fluorescence signal of ROS in the PBS-treated 4T1 cells, the fluorescence signal of ROS in the 4T1 cells treated with FA-CuS PENs@EGCG, FeCl3, CuS PENs@Fe-EGCG, FA-CuS PENs@Fe-EGCG, and FA-CuS PENs@Fe-EGCG + laser gradually increased (c), indicating that the oxidation-promoting effect of EGCG and the CDT effect induced by Fe(III) can produce a certain level of ROS in cells. Figure 18 c), and the relative ROS level in the cells was quantitatively analyzed, and the results are shown in Figure 18As shown in FIG. 9D, it can be found that the relative ROS levels in 4T1 cells treated with different materials are in the order of FA-CuS PENs@EGCG group < FeCl3 group < CuS PENs@Fe-EGCG group < FA-CuS PENs@Fe-EGCG group < FA-CuS PENs@Fe-EGCG+laser group. The relative ROS level in 4T1 cells treated with FA-CuS PENs@Fe-EGCG is significantly higher than that in 4T1 cells treated with FA-CuS PENs@EGCG (p<0.001), because Fe(III) is contained in FA-CuS PENs@Fe-EGCG compared with FA-CuS PENs@EGCG, and the released Fe(III) can induce ROS generation, while the released EGCG in FA-CuS PENs@Fe-EGCG not only promotes the conversion of Fe(III) to Fe(II) to accelerate the CDT process to generate ROS, but also generates ROS by itself, and the three synergies increase the intracellular ROS level. In addition, the relative ROS level in 4T1 cells treated with FA-CuS PENs@Fe-EGCG+laser is significantly higher than that in 4T1 cells treated with FA-CuS PENs@EGCG (p<0.001), because on the basis of Fe(III) and EGCG promoting ROS generation, the high temperature caused by CuS NPs-mediated PTT of laser-irradiated FA-CuS PENs@Fe-EGCG can also enhance the generation of Fe(III)-induced ROS, and the above synergies make FA-CuS PENs@Fe-EGCG+laser produce much higher ROS in 4T1 cells than FA-CuS PENs@EGCG. At the same time, the relative ROS level in 4T1 cells treated with FA-CuS PENs@Fe-EGCG is significantly higher than that in 4T1 cells treated with CuS PENs@Fe-EGCG (p<0.01), because the targeting effect of FA can increase the uptake of FA-CuS PENS@Fe-EGCG by 4T1 cells, and under the action of high-quantity-uptake Fe(III) and EGCG-induced intracellular ROS generation, the 4T1 cells treated with FA-CuS PENs@Fe-EGCG produce higher ROS than CuS PENs@Fe-EGCG.Finally, the relative ROS level in 4T1 cells treated with FA-CuS PENs@Fe-EGCG+laser was significantly higher than that in FA-CuS PENs@Fe-EGCG-treated 4T1 cells (p<0.001). This indicates that even though both FA-CuS PENs@Fe-EGCG and FA-CuS PENs@Fe-EGCG+laser exhibit Fe(III) and EGCG-induced ROS generation, the high temperature generated by the PTT mediated by CuS NPs in FA-CuS PENs@Fe-EGCG+laser-treated 4T1 cells enhances Fe(III)-induced ROS generation. Therefore, FA-CuS PENs@Fe-EGCG+laser exhibits the strongest ability to induce ROS generation in 4T1 cells among the materials analyzed above. This result is consistent with the apoptosis test results in Example 14, indicating that FA-CuS PENs@Fe-EGCG+laser induced 4T1 cells to produce a high amount of ROS, thereby leading to a large number of 4T1 cell apoptosis.

[0087] 15. Since upregulation of intracellular ROS levels induces the consumption of intracellular GSH, the GSH content in 4T1 cells treated with different materials was tested using a GSH / GSSG kit to investigate the ability of FA-CuS PENs@Fe-EGCG to consume intracellular GSH. First, at a concentration of 2.0 × 10⁻⁶... 5 The cell / well density was determined by seeding 4T1 cells in 12-well plates and culturing them overnight. The culture medium was then discarded, and the cells were incubated for 6 hours with media containing 0.32 mM CuS (FA-CuS PENs@EGCG, CuS PENs@Fe-EGCG, FA-CuSPENs@Fe-EGCG) and 80 μM FeCl3, respectively. The Fe concentration in the FeCl3-containing medium ([Fe] = 80 μM) was comparable to that in the CuS-containing media containing CuS PENs@Fe-EGCG and FA-CuS PENs@Fe-EGCG. PBS-treated 4T1 cells served as a control. After incubation, the culture medium was discarded, and the cells were washed 2-3 times with PBS. The 4T1 cells incubated with FA-CuS PENs@Fe-EGCG were divided into two groups: one group treated with laser (1064 nm, 1.0 W / cm²). 2, 5 min) irradiated cell groups and no laser irradiated cell groups. After the cells in each group were treated with / without laser irradiation, they were washed, digested, and centrifuged to obtain 6 groups of 4T1 cells treated differently, with 3 parallels in each group. Then, the standard operation procedures of the GSH / GSSG detection kit were followed for operation, and finally the absorbance value of the solution at 412 nm was determined using a microplate reader, and then the GSH content in the cells was calculated according to the absorbance value. The test results are as follows Figure 18As shown in Fig. 6e, the GSH content in 4T1 cells treated with different materials was ranked as follows: PBS group > FA-CuS PENs@EGCG group > FeCl3 group > CuS PENs@Fe-EGCG group > FA-CuS PENs@Fe-EGCG group > FA-CuS PENs@Fe-EGCG+laser group. Compared with the GSH content in 4T1 cells treated with PBS, the GSH content in 4T1 cells treated with FA-CuS PENs@EGCG was reduced, which might be due to the up-regulation of ROS caused by the pro-oxidation of EGCG in FA-CuS PENs@EGCG, leading to the consumption of intracellular GSH to maintain the intracellular redox homeostasis, thus reducing the GSH content. In addition, the GSH content in 4T1 cells treated with FA-CuS PENs@Fe-EGCG was significantly lower than that in 4T1 cells treated with FA-CuS PENs@EGCG (p<0.001), which was due to the presence of Fe(III) in FA-CuS PENs@Fe-EGCG, which could react with endogenous GSH in cells to induce CDT in cancer cells and consume more intracellular GSH. The GSH content in 4T1 cells treated with FA-CuS PENs@Fe-EGCG+laser was significantly lower than that in 4T1 cells treated with FA-CuS PENs@EGCG (p<0.001), which was due to the high amount of Fe(III) released by the temperature rise caused by PTT mediated by CuS NPs in FA-CuS PENs@Fe-EGCG under laser irradiation, which could consume more GSH to induce CDT in cancer cells, resulting in a significant reduction in intracellular GSH content. At the same time, the GSH content in 4T1 cells treated with FA-CuS PENs@Fe-EGCG was significantly lower than that in 4T1 cells treated with CuS PENs@Fe-EGCG (p<0.05), which was due to the targeting effect of FA, which increased the specific uptake of 4T1 cells to FA-CuS PENs@Fe-EGCG, and higher Fe(III) uptake promoted the consumption of intracellular GSH by FA-CuS PENs@Fe-EGCG to induce CDT in cancer cells.Finally, the GSH content in FA-CuS PENs@Fe-EGCG + laser treated 4T1 cells was significantly lower than that in FA-CuS PENs@Fe-EGCG treated 4T1 cells (p < 0.01), which was due to the fact that the elevated temperature caused by CuS NPs mediated PTT after laser irradiation of FA-CuS PENs@Fe-EGCG promoted the high release of Fe(III), and the high amount of Fe(III) consumed more GSH in the cells to induce CDT in cancer cells. Therefore, FA-CuS PENs@Fe-EGCG + laser had the strongest GSH consumption ability among the various groups of materials analyzed above.

[0088] 16、Cellular ROS content was up-regulated, and GSH content was down-regulated, indicating that the Fe(III)-based nanocomposites in the previous study might induce the production of LPO in cells. Therefore, the LPO content in 4T1 cells treated with different materials was explored. Again, each group of nanomaterials used unmodified FI to avoid the interference of the fluorescence of FI itself on the experimental results. First, 4T1 cells were seeded in a 12-well plate at a density of 2.0 x 105cells / well and cultured overnight, and then the culture medium was discarded and replaced with a culture medium containing CuS at a concentration of 0.32 mM, FA-CuS PENs@EGCG, CuS PENs@Fe-EGCG, FA-CuS PENs@Fe-EGCG, and FeCl3at a concentration of 80 μM, respectively, for 6 h. The culture medium containing FeCl3([Fe] = 80 μM) was equivalent to the Fe concentration in the culture medium containing CuS PENs@Fe-EGCG and FA-CuS PENs@Fe-EGCG with CuS at a concentration of 0.32 mM. At the same time, 4T1 cells treated with PBS were used as a control. After incubation, the culture medium was discarded, and the cells were washed with PBS for 2-3 times. The 4T1 cells incubated with FA-CuS PENs@Fe-EGCG were divided into two groups, i.e., laser (1064 nm, 1.0 W / cm2, 5 min) irradiated cell group and non-laser irradiated cell group. After the cells treated with each group of materials were treated with / without laser irradiation, C11-BODIPY 581 / 591 (lipid peroxidation probe diluted in serum-free medium) was added to each well and co-incubated for 20 min. Then, the cells in each group were washed, digested, and collected in a flow tube, and 3 parallel samples were prepared for each group for flow cytometry test. The results were analyzed by FlowJo software. 5 2 581 / 591 Figure 18 ​​​It can be seen that the relative LPO level in 4T1 cells treated with FA-CuS PENs@EGCG, FeCl3, CuSPENs@Fe-EGCG, FA-CuS PENs@Fe-EGCG and FA-CuS PENs@Fe-EGCG+laser gradually increased. By quantitatively analyzing the relative LPO level, it can be found that the relative LPO level in 4T1 cells treated with FA-CuS PENs@Fe-EGCG was significantly higher than that in 4T1 cells treated with FA-CuS PENs@EGCG (p<0.001). This is because compared with FA-CuS PENs@EGCG, FA-CuS PENs@Fe-EGCG contains Fe(III), and the released Fe(III) can induce the generation of ROS. At the same time, the released EGCG in FA-CuS PENs@Fe-EGCG not only promotes the conversion of Fe(III) to Fe(II) to accelerate the CDT process, but also produces ROS through its own pro-oxidation process. The high amount of ROS generated by the synergistic effect of the three oxidizes the lipid molecules on the cell membrane, triggering the lipid peroxidation chain reaction, resulting in a higher relative LPO level in 4T1 cells treated with FA-CuS PENs@Fe-EGCG than in 4T1 cells treated with FA-CuS PENs@EGCG. In addition, the relative LPO level in 4T1 cells treated with FA-CuS PENs@Fe-EGCG+laser was significantly higher than that in 4T1 cells treated with FA-CuS PENs@EGCG (p<0.001). This is because under the action of laser, the PTT mediated by CuS NPs in FA-CuS PENs@Fe-EGCG enhances the generation of ROS induced by Fe(III) due to the increase in temperature, and the ROS produced by the pro-oxidation effect of EGCG also synergistically induces FA-CuS PENs@Fe-EGCG+laser to produce more ROS in 4T1 cells. Excessive ROS oxidizes the lipid molecules on the cell membrane, ultimately leading to an increase in the relative LPO level in the cells.Meanwhile, the relative LPO level in FA-CuSPENs@Fe-EGCG treated 4T1 cells was significantly higher than that in CuSPENs@Fe-EGCG treated 4T1 cells (p<0.001), due to the fact that the targeting of FA increased the specific uptake of FA-CuSPENs@Fe-EGCG by 4T1 cells compared to non-targeted CuSPENs@Fe-EGCG treated 4T1 cells, the high uptake of Fe(III) and EGCG induced the generation of high amount of ROS in 4T1 cells, the high amount of ROS oxidized the lipid molecules on the cell membrane, triggered the lipid peroxidation chain reaction, and thus resulted in the higher relative LPO level in FA-CuSPENs@Fe-EGCG treated 4T1 cells than in CuSPENs@Fe-EGCG treated 4T1 cells. Finally, the relative LPO level in FA-CuSPENs@Fe-EGCG + laser treated 4T1 cells was 4.8, which was significantly higher than that in FA-CuSPENs@Fe-EGCG treated 4T1 cells (4.1, p<0.01), indicating that under laser irradiation, the high temperature caused by CuSNPs mediated PTT in FA-CuSPENs@Fe-EGCG enhanced the ROS generation induced by Fe(III), and the excessive ROS oxidized the lipid molecules on the cell membrane, thus resulting in the higher LPO in FA-CuSPENs@Fe-EGCG + laser treated 4T1 cells than in FA-CuSPENs@Fe-EGCG treated 4T1 cells. Figure 18 g) In summary, FA-CuSPENs@Fe-EGCG + laser could effectively generate ROS, deplete GSH, accumulate LPO, disrupt the redox homeostasis in cancer cells, and finally induce cancer cell apoptosis.

Claims

1. A Fe(III)-EGCG network-encapsulated copper sulfide nanocomposite, characterized in that, It is prepared by the following steps: Step 1: (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added to the dimethyl sulfoxide solution of folic acid for activation. Then, the solution is added to the dimethyl sulfoxide solution of amino-polyethylene glycol-carboxyl to react and obtain FA-PEG-COOH. Step 2: (3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are added to the aqueous solution of FA-PEG-COOH for activation, and then added to the aqueous solution of polyethyleneimine for reaction to obtain PEI.NH2-(PEG-FA); Step 3: Add the dimethyl sulfoxide solution of fluorescein isothiocyanate to the dimethyl sulfoxide solution of PEI.NH2-(PEG-FA), stir the reaction in the dark, and obtain PEI.NH2-(PEG-FA)-FI; Step 4: Add the aqueous solution of copper chloride dihydrate to the aqueous solution of PEI.NH2-(PEG-FA)-FI, stir in the dark, and then add the aqueous solution of sodium sulfide nonahydrate under heating conditions. Stir in the dark and wait for the temperature of the mixture to drop to room temperature. Then add triethylamine and stir in the dark to react. Finally, add acetic anhydride and continue to stir in the dark to react, and obtain FA-CuS PENs. Step 5: Add ferric chloride aqueous solution to the aqueous solution of FA-CuS PENs, stir and mix well, then add epigallocatechin gallate aqueous solution, stir and react to obtain the Fe(III)-EGCG network-encapsulated copper sulfide nanocomposite FA-CuS PENs@Fe-EGCG.

2. The Fe(III)-EGCG network-encapsulated copper sulfide nanocomposite according to claim 1, characterized in that, In step 1, the molar ratio of (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and folic acid is 1-1.5:1-1.5:1, and the molar ratio of folic acid to amino-polyethylene glycol-carboxyl group is 1.2-1.5:

1.

3. The Fe(III)-EGCG network-encapsulated copper sulfide nanocomposite according to claim 1, characterized in that, In step 2, the molar ratio of (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and FA-PEG-COOH is 10-12:10-12:1, and the molar ratio of FA-PEG-COOH and polyethyleneimine is 20-25:

1.

4. The Fe(III)-EGCG network-encapsulated copper sulfide nanocomposite according to claim 1, characterized in that, In step 3, the molar ratio of fluorescein isothiocyanate to PEI.NH2-(PEG-FA) is 3-5:

1.

5. The Fe(III)-EGCG network-encapsulated copper sulfide nanocomposite according to claim 1, characterized in that, In step 4, the molar ratio of copper chloride dihydrate to PEI.NH2-(PEG-FA)-FI is 50-52:1, the molar ratio of sodium sulfide nonahydrate to copper chloride dihydrate is 3:1, and the molar ratio of triethylamine, acetic anhydride to PEI.NH2-(PEG-FA)-FI is 120-660:100-550:

1.

6. The Fe(III)-EGCG network-encapsulated copper sulfide nanocomposite according to claim 1, characterized in that, In step 4, the heating temperature is 70℃.

7. The Fe(III)-EGCG network-encapsulated copper sulfide nanocomposite according to claim 1, characterized in that, In step 5, the molar ratio of ferric chloride to FA-CuS PENs is 10-12:1, and the molar ratio of epigallocatechin gallate to FeCl3 is 2:

1.

8. The use of the Fe(III)-EGCG network-encapsulated copper sulfide nanocomposite according to any one of claims 1 to 7 in the preparation of tumor diagnostic and therapeutic products.

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