Application of chlorin-loaded rare earth up-conversion nanoparticles and anti-tumor and anti-inflammatory drug

Abstract

The invention relates to application of chlorin-loaded rare earth upconversion nanoparticles and an anti-tumor and anti-inflammatory drug, and relates to the field of biological medicines. The chlorin-loaded rare earth up-conversion nanoparticles are used for preparing anti-tumor or / and anti-inflammatory drugs. The chlorin-loaded rare earth up-conversion nanoparticles disclosed by the invention do not generate toxicity to normal cells in a concentration range of 31.25-500mu g / ml, and can effectively inhibit migration of tumor cells and inflammation related mechanisms; the tumor targeting agent has no toxicity to mice within 120 mg / kg in mice, can target tumor sites and reach lungs through blood circulation, and 20 mg / kg of the tumor targeting agent can significantly inhibit tumor volume and tumor pulmonary metastasis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of dihydroporphyrin rare earth upconversion nanoparticles and antitumor and anti-inflammatory drugs. Background Technology

[0002] Currently, over 90% of cancer-related deaths are caused by tumor cell metastasis. Most tumor cell metastasis is related to chronically high inflammation levels, and chronic inflammation has become one of the important causes in the metastasis process [Deretic V. Autophagy in inflammation, infection, and immunometabolism[J]. Immunity, 2021, 54(3):437-453.]. Chronic inflammation and cytokine storms are uncontrolled inflammatory responses [Hirano T. IL-6 inflammation, autoimmunity and cancer. Int Immunol. 2021 Mar 1; 33(3):127-148.]. When the body is stimulated by microorganisms or drugs, the production of various cytokines increases sharply, which can lead to an imbalance in the immune system and is a serious and life-threatening syndrome [Jarczak D, Nierhaus A. Cytokine Storm-Definition, Causes, and Implications[J]. Int J Mol Sci, 2022, 23(19):11740.]. Once activated, the death pathways of pyroptosis and programmed cell necrosis can induce a strong inflammatory response in vivo, leading to pathological changes in various inflammatory diseases [Weinlich R, Oberst A, Beere HM et al. Necroptosis in development, inflammation and disease[J]. Nat Rev Mol Cell Biol, 2017, 18: 127-36. and Liu X, Lieberman J. A mechanism understanding of pyroptosis: the fiery death triggered by invasive infection[J]. Adv Immunol, 2017, 135: 81-117.]. The classic pathway of pyroptosis involves Caspase 1 cleavage and activation of the GSDMD gene, which forms pores, thereby exposing the N-terminal region and releasing pro-inflammatory cytokines IL-18 and IL-1β to signal cell death [De Miguel D, Ramirez-Labrada A, Uranga I, et al. Inflammatory cell death induced by cytotoxic lymphocytes: a dangerous but necessary liaison[J]. FEBS J, 2022, 289(15): 4398-4415.].The classic pathway of programmed cell death (PCD) involves RIPK3-mediated phosphorylation of MLKL, the executor of PCD, triggering MLKL oligomerization and membrane binding, leading to plasma membrane damage and the release of potential inflammatory factors [Frank D, Vince JE. Pyroptosis versus necroptosis: similarities, differences, and crosstalk[J]. Cell Death Differ. 2019, 26(1): 99-114.]. Immunogenic cell death (ICD) is also associated with inflammation, and its activation produces a series of DAMPs that induce an immune response [Qin X, Liu J, Xu Y, et al. Mesoporous Bi-Containing Radiosensitizer Loading with DOX to Repolarize Tumor-Associated Macrophages and Elicit Immunogenic Tumor Cell Death to Inhibit Tumor Progression[J]. ACS Appl Mater Interfaces, 2020, 12(28): 31225-31234.]. The report states that [Xu Z, Xu M, Wu X, et al. A Half-Sandwich Ruthenium(II)(N^N) Complex: Inducing Immunogenic Melanoma Cell Death in Vitro and in Vivo[J]. ChemMedChem, 2023, 18(16): e202300131.] that the process of ICD not only causes the exposure of CRT and HSP70 proteins, but also reduces the total intracellular protein. Programmed cell death (PCD) can induce various immune responses and inflammatory factors, among which pyroptosis, programmed cell necrosis, and ICD are all cell death mechanisms related to inflammatory responses [Qiao H, Cui Z, Yang S, et al. Targeting Osteocytes to Attenuate Early Breast Cancer Bone Metastasis by Theranostic Upconversion Nanoparticles with Responsive Plumbagin Release[J]. ACS Nano, 2017, 11(7): 7259-7273.].

[0003] Existing photosensitizers have no inhibitory effect on tumor cells when used at safe doses without photoexcitation; furthermore, the inhibitory effect of dihydroporphyrin rare-earth upconversion nanoparticles on tumor cell migration and inflammation-related mechanisms without laser excitation has not been reported. Therefore, this invention provides applications of dihydroporphyrin rare-earth upconversion nanoparticles and antitumor and anti-inflammatory drugs. Summary of the Invention

[0004] The technical problem to be solved by this invention is to provide applications of dihydroporphyrin rare-earth upconversion nanoparticles and anti-tumor and anti-inflammatory drugs. The aim is to systematically elucidate the role of dihydroporphyrin rare-earth upconversion nanoparticles in inhibiting migration and inflammation-related mechanisms in tumor cells, hoping to provide new insights into the research of nanoparticle anti-tumor mechanisms.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: The first aspect is the application of dihydroporphyrin rare earth upconversion nanoparticles, which are used in the preparation of antitumor and / or anti-inflammatory drugs.

[0006] Among them, the dihydroporphyrin rare earth upconversion nanoparticles (referred to as UCNPs@Ce6) were prepared according to the method described in patent publication number CN118792051A, entitled "A Method for Preparing and Applying Dihydroporphyrin Rare Earth Upconversion Nanoparticles." The specific structure of the dihydroporphyrin rare earth upconversion nanoparticles is: NaYF4:Er@NaYbF4@NaYF4@Ce6; the dihydroporphyrin rare earth upconversion nanoparticles are prepared by combining rare earth upconversion nanoparticles and dihydroporphyrin and modifying them with polyethylene glycol; the rare earth upconversion nanoparticles, from the inside out, include: a core layer, a first shell layer, and a second shell layer, wherein the core layer, the first shell layer, and the second shell layer are NaYF4:Er nanoparticles, NaYbF4, and NaYF4, respectively; the rare earth upconversion nanoparticles produce the highest fluorescence emission peak at 650-670 nm when excited by near-infrared light at 980 nm.

[0007] Furthermore, the tumor includes at least one of esophageal squamous cell carcinoma and breast cancer.

[0008] Furthermore, the application of dihydroporphyrin rare earth upconversion nanoparticles is characterized in that the dihydroporphyrin rare earth upconversion nanoparticles exert anti-tumor effects by inhibiting tumor growth volume, tumor cell lung metastasis, and tumor inflammatory response.

[0009] Furthermore, the loaded dihydroporphyrin rare-earth upconversion nanoparticles exert anti-inflammatory effects by inhibiting tumor inflammation-related pathways.

[0010] In a second aspect, an antitumor and / or anti-inflammatory drug, said drug comprising the loaded dihydroporphyrin rare earth upconversion nanoparticles in the application of the aforementioned loaded dihydroporphyrin rare earth upconversion nanoparticles.

[0011] The method for preparing the antitumor and / or anti-inflammatory drugs of the present invention can be achieved using conventional preparation methods.

[0012] Furthermore, the drug also includes pharmaceutically acceptable carriers and / or auxiliary components.

[0013] Furthermore, the drug also includes at least one of the following: filler, lubricant, dispersant, wetting agent, binder, regulator, solubilizer, antioxidant, antibacterial agent, emulsifier, and disintegrant.

[0014] Furthermore, the dosage form of the drug is a clinically acceptable pharmaceutical preparation.

[0015] Furthermore, the dosage form of the drug includes any one of capsules, granules, tablets, pills, syrups, powders, granules, suppositories, drops, and emulsions.

[0016] The actual dosage of the active ingredient in the drug of the present invention (the loaded dihydroporphyrin rare earth upconversion nanoparticles of the present invention) should be determined based on a variety of relevant factors, including the severity of the disease to be treated, the route of administration, the patient's age, gender, and weight. Therefore, the above dosage should not limit the scope of protection of the present invention in any way.

[0017] The dihydroporphyrin rare-earth upconversion nanoparticles mentioned in this invention were subjected to the following verification experiments: (1) The dihydroporphyrin rare earth upconversion nanoparticles of the present invention are non-toxic to human esophageal normal epithelial cells (HEEC) and human esophageal squamous cell carcinoma cells (EC109) in the concentration range of 31.25-500 μg / ml. A pharmacodynamic study was conducted using 250 μg / ml of the dihydroporphyrin rare earth upconversion nanoparticles of the present invention, and it was found that 250 μg / ml of the dihydroporphyrin rare earth upconversion nanoparticles significantly inhibited EC109 cell migration. EC109 cells treated with 250 μg / ml dihydroporphyrin rare-earth upconversion nanoparticles were used to collect cell supernatants. The release of pyroptosis-related inflammatory cytokine IL-1β, programmed necrosis-related inflammatory cytokine TNF-α, and immunogenic cell death (ICD)-related inflammatory cytokine HMGB1 was detected using a double-sandwich ELISA assay. Western blotting was used to detect the expression levels of pyroptosis-related proteins Caspase1, GSDMD, and Cleaved GSDMD-N; programmed necrosis-related proteins MLKL, RIP3, p-MLKL, and p-RIP3; and ICD-related proteins CRT and HSP70. The results showed that treatment with 250 μg / ml dihydroporphyrin rare-earth upconversion nanoparticles reduced the levels of pyroptosis-related proteins Caspase1, GSDMD, and Cleaved GSDMD-N. The expression levels of GSDMD-N, a rare-earth upconversion nanoparticle, including the proteins MLKL, RIP3, p-MLKL, and p-RIP3 associated with programmed necrosis (ICD), had no effect on the expression levels of the immunogenic cell death-associated proteins CRT and HSP70. Reports indicate that ICD not only causes exposure of CRT and HSP70 proteins but also reduces total intracellular protein levels. The fact that ICD-associated proteins CRT and HSP70 did not decrease suggests that the ICD process was inhibited. Based on these results, it was found that dihydroporphyrin rare-earth upconversion nanoparticles can inhibit inflammation-related mechanisms.

[0018] (2) Further verification of the effect of dihydroporphyrin rare earth upconversion nanoparticles on inhibiting tumor cell metastasis at a safe dose was conducted in animal experiments. A xenograft model was established using mouse breast cancer cells (4T1) with high invasiveness and tumorigenicity. Dihydroporphyrin rare earth upconversion nanoparticles at a concentration of 120 mg / kg were injected into mice via the tail vein. No toxicity was observed in the mice. Subsequently, a one-step efficacy verification was performed at a concentration of 20 mg / ml. It was found that the nanoparticles could target the tumor site and reach the lungs through blood circulation, and could significantly inhibit tumor volume and lung metastasis of cancer cells.

[0019] The beneficial effects of this invention are: the dihydroporphyrin rare earth upconversion nanoparticles of this invention do not produce toxicity to normal cells in the concentration range of 31.25-500 μg / ml, and can effectively inhibit the migration of tumor cells and tumor inflammation-related mechanisms; in mice, the concentration of up to 120 mg / kg does not produce toxicity, can target the tumor site, and can reach the lungs through blood circulation; 20 mg / kg can significantly inhibit tumor volume and tumor lung metastasis. Attached Figure Description

[0020] Figure 1 The figure shows the safety test results of UCNPs@Ce6 of the present invention on EC109 and HEEC cells. (A) Cell survival rate of the two cells after co-culturing with different concentrations of UCNPs@Ce6 for 24 h; (B) Cell survival rate of the two cells after co-culturing with different concentrations of UCNPs@Ce6 for 48 h. Figure 2 The diagram shows the inhibition of EC109 cell migration by UCNPs@Ce6 according to the present invention, wherein (A) is a representative diagram of the inhibition of EC109 cell migration by UCNPs@Ce6 at different time points, and (B) is a statistical diagram of the inhibition of EC109 cell migration rate by UCNPs@Ce6; compared with Blank, **P<0.01; Figure 3 This is a diagram showing the inhibition of inflammatory factor release by UCNPs@Ce6 in this invention; wherein, (AB) analysis of the content of pyroptosis-related inflammatory factors IL-18 and IL-1β; (C) analysis of the content of TNF-α, a cell programmed necrosis-related inflammatory factor; (D) analysis of the immunogenic cell death-related inflammatory factor HMGB1; compared with Blank, **P<0.01, ***P<0.0001; Figure 4 This is a protein expression diagram of the tumor inflammation-related mechanism inhibited by UCNPs@Ce6 of the present invention. (A) Representative Western blot analysis of proteins related to programmed cell death, pyroptosis, and immunogenic cell death after 24 hours of treatment with different methods on EC109 cells; (BD) Relative expression of programmed cell death-related proteins MLKL, p-MLKL, RIP3, and p-RIP3 after 24 hours of treatment with different methods on EC109 cells; (EG) Relative expression of pyroptosis-related proteins Cleaved GSDMD-N, Caspase1, and GSDMD after 24 hours of treatment with different methods on EC109 cells; (HJ) Relative expression of immunogenic cell death-related proteins CAT and HSP70 after 24 hours of treatment with different methods on EC109 cells. Compared with Blank, *P<0.05, **P<0.01, ***P<0.0001; Figure 5The UCNPs@Ce6 of the present invention can target tumors and lungs; wherein, (A) in vivo fluorescence imaging 6 hours after UCNPs@Ce6 injection, (B) ex vivo fluorescence imaging 6 hours after UCNPs@Ce6 injection; Figure 6 The safety profile of UCNPs@Ce6 of the present invention in mice is shown in the figure, where (A) mouse body weight growth curve; (B) heart index; (C) liver index; (D) spleen index; (E) lung index; (F) kidney index; (G) alanine aminotransferase level; (H) aspartate aminotransferase level; (I) urea level; (J) creatinine level; (K) HE staining of heart, liver, spleen, lung and kidney sections, n=6; Figure 7 The UCNPs@Ce6 of this invention can inhibit tumor volume and lung metastasis. The results include: (A) representative tumors of each group; (B) whole-body tumor images of mice; (C) average tumor growth curve; (D) tumor weight and tumor inhibition rate results; and (E) statistical graph of the number of lung tumors. Detailed Implementation

[0021] The principles and features of this invention are described below. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0022] Description of the source of materials and reagents: Dihydroporphyrin rare earth upconversion nanoparticles (referred to as UCNPs@Ce6) were prepared by the following method: (1) Preparation of NaYF4:Er nanoparticles: 1 ml of aqueous solution containing YCl3·6H2O and ErCl3·6H2O with a total rare earth ion content of 0.5 mmol was added to a three-necked flask (50 ml). After the water was completely evaporated, 3.75 ml of oleic acid (OA) was added first, followed by 7.5 ml of 1-octadecene (ODE). The mixture was heated to 120°C under argon protection and held for 30 minutes. Then, the system temperature was raised to 160°C and reacted for 60 minutes. 5 ml of methanol solution containing 0.05 g NaOH and 0.074 g NH4F was added under stirring. The mixture was stirred vigorously at room temperature for 30 minutes. Subsequently, the system temperature was raised to 100°C under argon conditions and reacted for 30 minutes. The water and methanol solution in the reaction mixture were then removed. Under argon protection, with a condenser added, the temperature was rapidly raised to 300℃ and maintained for 90 minutes. The reaction was then stopped after completion. The reaction system was allowed to cool naturally to room temperature, washed several times with cyclohexane and ethanol, and dispersed in 5 ml of cyclohexane for later use.

[0023] (2) Preparation of NaYF4:Er@NaYbF4 nanoparticles: Add 1 ml of aqueous solution containing YbCl3·6H2O with a total rare earth ion content of 1.5 mmol to a 50 ml flask. The method is the same as above, except that 7.5 ml of OA and 15 ml of ODE are added. After the methanol solution is added, the NaYF4:Er previously dispersed in cyclohexane is added dropwise to the reaction solution.

[0024] (3) Preparation of NaYF4:Er@NaYbF4@NaYF4 nanoparticles: Add 1 ml of aqueous solution containing YCl3·6H2O and a total rare earth ion content of 1.2 mmol to a 50 ml flask. The method is the same as above, except that 7.5 ml of OA and 15 ml of ODE are added. After the methanol solution is added, the previously dispersed NaYF4:Er@NaYbF4 in cyclohexane is added dropwise to the reaction solution.

[0025] (4) Modification: 50 mg DSPE-PEG 2000 (distearylphosphatidylethanolamine-polyethylene glycol 2000) and 10 mg Ce6 were dissolved in 5 ml of chloroform, and the chloroform solution containing rare earth upconversion nanoparticles with a concentration of 1 mg / ml was added. After ultrasonic dispersion, 5 ml of ultrapure water was added to the mixture, and then the mixture was rotary evaporated to remove chloroform. After ultrasonic dispersion and cooling to room temperature, the mixture was filtered through a 0.22±0.1 μm filter membrane, and the filtrate was centrifuged, washed with purified water more than six times, and then freeze-dried into powder using a freeze dryer and stored at -20℃ for later use.

[0026] EC109 cells; HEEC cells; CCK8 working solution; RPMI 1640 medium; 4T1 cells; BALB / c-nu nude mice.

[0027] EC109 cells were purchased from Guangzhou Saiku Biotechnology Co., Ltd.; HEEC cells were purchased from Guangzhou Genio Biotechnology Co., Ltd.; 4T1 cells were purchased from the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences; RPMI 1640 and MEM media were from Gibco, USA; nude mice were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd.

[0028] Example 1 1. Experimental methods.

[0029] 1.1 Cell safety testing.

[0030] Safety testing: EC109 and HEEC cells were cultured until they were in good condition, then digested and collected. The concentration was adjusted to 80,000 / ml. 100 μL was added to each well of a 96-well plate. After 24 hours of cell adhesion, the plates were seeded for 24 hours. The medium was then replaced with complete medium containing 500 μg / ml, 250 μg / ml, 125 μg / ml, 62.5 μg / ml, and 31.25 μg / ml UCNPs@Ce6. The cells were cultured for another 24 hours or 48 hours. The CCK8 working solution was prepared according to the ratio of complete medium to CCK8 reagent = 9:1.

[0031] For the two cell lines described above, the culture medium was aspirated, and the cells were washed once with PBS. 100 μL of LCK8 working solution was added to each well, and the cells were incubated in a cell culture incubator for 1 hour. The OD value at 450 nm was then measured using a microplate reader. Cell viability was calculated according to the following formula (1): (1); 1.2 Scratch test.

[0032] Scratch assay: EC109 cells were diluted to 250,000 / ml, and 2ml was seeded into a 35mm culture dish. After the cells reached confluence under the above conditions for 24 hours, a 200µL pipette tip was used to scribble a line perpendicular to the bottom of the well plate to form an intersection point for easy observation. The cells were washed three times with serum-free medium to remove the scribbled cell clumps. The first time point was 0h, and the drug was administered immediately after the scratch. The second time point was 24h after the drug was administered and 980nm laser irradiation was performed. The third time point was 48h, after photodynamic therapy and incubation for another 24h.

[0033] According to the experimental plan, the Only UCNPs@Ce6 group was immediately given 2 ml of RPMI 1640 medium containing 2% FBS and 250 ug / ml UCNPs@Ce6 after scratching, while the Blank group was given 2 ml of RPMI 1640 medium containing 2% FBS and cultured for 24 h. Scratch images of the same area were taken after scratching, 24 h after drug administration, and 24 h after treatment. The wound healing status was compared based on the scratch area, and the cell migration rate was calculated according to the following formula (2): (2); 1.3 Detection of inflammatory factors.

[0034] Inflammatory factor detection: A standard curve was established according to the instructions. Cell culture supernatant was collected, and the OD value was detected using an ELISA reader. The concentrations of IL-18, IL-1β, HMGB1, and TNF-α in the cell culture medium were obtained by subtracting the OD value from the standard curve.

[0035] 1.4 In vivo validation experiment in mice.

[0036] In vivo validation experiment in mice: 4T1 cells were prepared at a concentration of 1×10⁻⁶ using physiological saline. 7 A BALB / c-nu nude mouse model of breast cancer xenograft was established by injecting 0.1 ml of the xenograft into the right axilla of each of 10 nude mice. After tumor formation, the tumor diameter was measured using calipers to monitor tumor growth, which reached approximately 50 mm. 3 The animals were randomly divided into two groups: the Blank group and the Only UCNPS@Ce6 group, with 5 animals in each group.

[0037] 1.5 Fluorescence imaging.

[0038] Fluorescence imaging: 200uLUCNPS@Ce6 was injected via the tail vein. In vivo fluorescence imaging was performed 6 hours after injection. The mice were then sacrificed, and the lungs and tumors were removed for ex vivo fluorescence imaging.

[0039] 1.6 Validity test.

[0040] Effectiveness test: After the tumor volume reached the target, the mice were randomly divided into two groups: Blank group and Only UCNPs@Ce6 group. The Blank group was injected with saline via the tail vein every other day, and the Only UCNPs@Ce6 group was injected with UCNPS@Ce6 (20mg / kg). The experimental period was 15 days, and a total of 8 administrations and laser treatments were performed. The tumor volume was recorded every 2 days during the period. The tumor volume (V) was calculated according to the following formula (3), where “a” and “b” represent the longest and shortest values ​​of the tumor size, respectively. (3); The relative tumor volume (RTV) is calculated based on the measurement results. The calculation formula (4) is as follows, where "V0" is the volume of the tumor when the drug is administered in separate cages (i.e., d). 0 The tumor volume obtained by measurement, V t This represents the tumor volume at each measurement.

[0041] (4); Nude mice were euthanized immediately after treatment, and tumor masses were surgically removed and weighed. The tumor growth inhibition rate was calculated based on the tumor mass weight to evaluate the tumor-inhibiting effect of UCNPS@Ce6 in photodynamic therapy in nude mice. The calculation formula (5) is as follows: (5); 1.7 Animal safety testing.

[0042] (1) Animal safety dose range testing: Animals were randomly assigned to groups according to the dosing regimen, and the safety response of the test samples was dynamically observed after tail vein administration. Changes (activity, appearance, mental state) were observed during and after the administration process. After one week of continuous administration, the animals were kept alive and their condition was continuously observed to determine whether chronic toxicity would develop.

[0043] On the first day, administer a low dose of 5 mg / kg, a medium dose of 10 mg / kg, and a high dose of 20 mg / kg. On the second day, administer the low dose of 5 mg / kg, the medium dose of 10 mg / kg, and the high dose of 20 mg / kg. On the third day, administer the low dose of 10 mg / kg, the medium dose of 20 mg / kg, and the high dose of 40 mg / kg. On the fourth day, administer the low dose of 10 mg / kg, the medium dose of 20 mg / kg, and the high dose of 40 mg / kg. Observe the animals for 3 days.

[0044] On the eighth day, administer the low dose of 30 mg / kg, the medium dose of 60 mg / kg, and the high dose of 120 mg / kg. On the ninth day, administer the low dose of 30 mg / kg, the medium dose of 60 mg / kg, and the high dose of 120 mg / kg. The animals' condition will be observed for the next 7 days.

[0045] (2) Body weight and organ index: Animal weight was recorded every 2 days during treatment. Nude mice were euthanized immediately after treatment. Heart, liver, spleen, and lungs were surgically removed and weighed. The safety of UCNPS@Ce6 in nude mice was evaluated by organ index to determine whether it had any effect on major organs. Organ index was calculated according to the following formula (6): (6); (3) Serum biochemical analysis: Blood was collected when nude mice were euthanized, and liver function indicators (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) and kidney function indicators (urea (URE) and creatinine (CRE-S)) were analyzed using a fully automated blood biochemistry analyzer.

[0046] (4) HE staining: The heart, liver, spleen, lung and kidney tissues fixed in 10% formalin were prepared into paraffin sections. The sections were dewaxed and hydrated according to the standard method. The sections were soaked in xylene for 5 minutes, and then soaked in fresh xylene for another 5 minutes. Then, they were soaked in anhydrous ethanol, 95% ethanol, 85% ethanol and 70% ethanol for 5 minutes each in sequence. Finally, they were washed with PBS for 3 minutes each time.

[0047] According to the instructions of the HE staining kit, immerse the slide in the staining solution of reagent 1 for 5 min, wash with water for 60 s, immerse in dichromatic solution I for about 20 s, wash with water for about 60 s, immerse in trichromatic solution II for about 40 s, wash with water for about 60 s, and finally place it in the reagent dead staining solution for 2 min, then wash away the excess staining solution with reagent 5, wash twice, air dry, mount and examine under a microscope.

[0048] 2. Experimental results.

[0049] To investigate the effects of different concentrations on the growth viability of EC109 and HEEC cells, EC109 and HEEC cells were co-cultured with UCNPs@Ce6 at concentration gradients ranging from 31.25 μg / ml to 500 μg / ml for 24 h or 48 h. Cell viability was assessed using the CCK8 assay. Results are as follows: Figure 1 As shown in Figure AB, for both cell types, there were no significant differences between the groups and the Blank group after co-culturing with UCNPs@Ce6 at concentration gradients of 31.25 ug / ml-500 ug / ml for 24 h and 48 h (P>0.05). The results indicate that incubation with UCNPs@Ce6 for 24 h-48 h had no effect on the viability of EC109 and HEEC cells.

[0050] To investigate the inhibitory effect of UCNPs@Ce6 on EC109 cell migration, the first time point (0h) was set up after scratching and photographing. Drug administration was performed immediately after photographing, and incubation for 24h was the second time point. A further 24h incubation was the third time point (48h). Results are as follows: Figure 2 As shown in A to B, compared with the Blank group, the Only UCNPs@Ce6 group significantly inhibited EC109 cell migration (P < 0.05). The results suggest that using UCNPs@Ce6 alone can effectively inhibit EC109 cell migration.

[0051] EC109 cell supernatants after different treatments were collected, and the release of pyroptosis-related inflammatory cytokines IL-18 and IL-1β, programmed necrosis-related inflammatory cytokines TNF-α, and immunogenic cell death-related inflammatory cytokines HMGB1 were detected using a double-sandwich ELISA method. Results are as follows: Figure 3 As shown in A to D, compared with the Blank group, the Only UCNPs@Ce6 group significantly inhibited IL-1β, HMGB1, The release of TNF-α did not increase the release of IL-18, indicating that the presence of UCNPs@Ce6 could inhibit the release of inflammatory factors associated with pyroptosis, immunogenic cell death, and necrosis.

[0052] Pyroptosis, programmed necrosis, and immunogenic cell death are all cell death mechanisms related to tumor inflammation. Based on the ELISA results above, UCNPs@Ce6 can inhibit inflammatory factors involved in these cell death mechanisms; the results are as follows... Figure 4 As shown in A to J, compared with the Blank group, the Only UCNPs@Ce6 group significantly reduced the expression levels of programmed cell death-related proteins MLKL, RIP3, p-MLKL, p-RIP3, pyroptosis-related proteins Caspase1, GSDMD, and Cleaved GSDMD-N (All P < 0.05). None of the groups had any effect on immunogenic cell death-related proteins (P > 0.05). The results indicate that UCNPs@Ce6 can inhibit the expression of proteins related to cellular inflammation mechanisms.

[0053] To evaluate the targeting effect of UCNPs@Ce6 in vivo, 200 μl of UCNPs@Ce6 was injected intravenously into BALB / c-nu mice. In vivo fluorescence imaging was performed 6 hours later. The results are as follows: Figure 5 As shown in A to B, UCNPs@Ce6 was successfully enriched at the tumor site. After euthanizing the mice, the lungs and tumors were removed for in vitro fluorescence imaging. It was found that UCNPs@Ce6 may be due to the fluorescence of blood rings in the lungs.

[0054] In the in vivo safety study of UCNPs@Ce6 with periodically increased dosage, observations were made based on animal weight, appearance, mental state, and activity level. At dosages below 120 mg / kg, no significant changes were observed in animal condition compared to control mice; weight remained steadily increasing, activity range did not decrease, food and water intake did not decrease, and mental state remained unchanged. At a dosage of 120 mg / kg, slight changes in respiration were observed in the treated mice compared to control mice; no lethargy was observed, but the startle reflex was more pronounced. On the first day of administration, weight gain was slower than in control mice, and food intake was relatively reduced. Subsequently, slower weight gain and no change in food intake were observed in the treated group compared to the control group. Further validation of efficacy and safety was achieved at a dosage of 20 mg / kg. Nude mice were weighed every two days during the treatment period. No abnormal weight loss was observed during this period. After treatment, mice were euthanized by cervical dislocation, and the heart, liver, spleen, lungs, and kidneys were surgically removed and weighed. Organ indices were calculated using a formula, such as... Figure 6As shown in A to K, compared with the Blank group, there was no significant difference in the Only UCNPs@Ce6 group (P>0.05), and no abnormal decrease in organ indices was observed. Serum biochemical analysis of liver function indicators (ALT, AST) and kidney function indicators (URE, CRE-S) showed no significant differences among the groups compared with the Blank group (P>0.05), and no abnormal increase in biochemical indicators was observed. This indicates that UCNPs@Ce6 at a concentration of 20 mg / kg does not cause toxicity in nude mice after entering the body, and has good safety.

[0055] To investigate the in vivo antitumor effect of UCNPS@Ce6, a nude mouse model of breast cancer xenograft was established in BALB / c-nu mice. When the tumor volume reached 100 mm... 3 Mice were randomly divided into two groups: the Blank group received 200 μL of saline via tail vein injection every two days, and the Only UCNPs@Ce6 group received 200 μL of UCNPs@Ce6 via tail vein injection every two days. The results are as follows: Figure 7 As shown, compared with the Blank group, the Only UCNPs@Ce6 group significantly inhibited tumor volume growth (P<0.05) and the number of lung metastases from breast cancer (P<0.05).

[0056] In summary, (1) the dihydroporphyrin rare earth upconversion nanoparticles of the present invention are non-toxic to human esophageal normal epithelial cells (HEEC) and human esophageal squamous cell carcinoma cells (EC109) in the concentration range of 31.25 μg / ml to 500 μg / ml. A pharmacodynamic study was conducted using 250 μg / ml of the dihydroporphyrin rare earth upconversion nanoparticles of the present invention, and it was found that 250 μg / ml of the dihydroporphyrin rare earth upconversion nanoparticles significantly inhibited EC109 cell migration. EC109 cells treated with 250 μg / ml dihydroporphyrin rare-earth upconversion nanoparticles were used to collect cell supernatants. The release of pyroptosis-related inflammatory factors IL-18 and IL-1β, programmed necrosis-related inflammatory factor TNF-α, and immunogenic cell death (ICD)-related inflammatory factor HMGB1 were detected using a double-sandwich ELISA assay. Western blotting was used to detect the expression levels of pyroptosis-related proteins Caspase1, GSDMD, and Cleaved GSDMD-N; programmed necrosis-related proteins MLKL, RIP3, p-MLKL, and p-RIP3; and ICD-related proteins CRT and HSP70. The results showed that treatment with 250 μg / ml dihydroporphyrin rare-earth upconversion nanoparticles reduced the levels of pyroptosis-related proteins Caspase1, GSDMD, and Cleaved GSDMD-N. The expression levels of GSDMD-N, a rare-earth upconversion nanoparticle, including the proteins MLKL, RIP3, p-MLKL, and p-RIP3 associated with programmed necrosis (ICD), had no effect on the expression levels of the immunogenic cell death-associated proteins CRT and HSP70. Reports indicate that ICD not only causes exposure of CRT and HSP70 proteins but also reduces total intracellular protein levels. The fact that ICD-associated proteins CRT and HSP70 did not decrease suggests that the ICD process was inhibited. Based on these results, it was found that dihydroporphyrin rare-earth upconversion nanoparticles can inhibit inflammation-related mechanisms.

[0057] (2) Further verification of the effect of dihydroporphyrin rare earth upconversion nanoparticles on inhibiting tumor cell metastasis at a safe dose was conducted in animal experiments. A xenograft tumor model was established using mouse breast cancer cells (4T1) with high invasiveness and tumorigenicity. Dihydroporphyrin rare earth upconversion nanoparticles at a concentration of 120 mg / kg were injected into mice via the tail vein. No toxicity was observed in the mice. Subsequently, a one-step efficacy verification was conducted at a concentration of 20 mg / ml. It was found that the nanoparticles could target the tumor site and reach the lungs through blood circulation, and could significantly inhibit tumor volume and lung metastasis of cancer cells.

[0058] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The application of dihydroporphyrin rare-earth upconversion nanoparticles, characterized in that, Loaded dihydroporphyrin rare earth upconversion nanoparticles are used in the preparation of antitumor and / or anti-inflammatory drugs.

2. The application of the dihydroporphyrin rare-earth upconversion nanoparticles according to claim 1, characterized in that, The tumor includes at least one of esophageal squamous cell carcinoma and breast cancer.

3. The application of the dihydroporphyrin rare-earth upconversion nanoparticles according to claim 1, characterized in that, The loaded dihydroporphyrin rare earth upconversion nanoparticles exert anti-tumor effects by inhibiting tumor growth volume, tumor cell lung metastasis, and tumor inflammatory response.

4. The application of the dihydroporphyrin rare-earth upconversion nanoparticles according to claim 1, characterized in that, The loaded dihydroporphyrin rare-earth upconversion nanoparticles exert anti-inflammatory effects by inhibiting tumor inflammation-related pathways.

5. A drug for treating tumors and / or inflammation, characterized in that, The drug comprises the loaded dihydroporphyrin rare earth upconversion nanoparticles in the application of any one of claims 1 to 4.

6. The antitumor and / or anti-inflammatory drug according to claim 5, characterized in that, The drug also includes pharmaceutically acceptable carriers and / or auxiliary components.

7. The antitumor and / or anti-inflammatory drug according to claim 5, characterized in that, The drug also includes at least one of the following: filler, lubricant, dispersant, wetting agent, binder, regulator, solubilizer, antioxidant, antibacterial agent, emulsifier, and disintegrant.

8. The antitumor and / or anti-inflammatory drug according to claim 5, characterized in that, The dosage form of the drug is a clinically acceptable pharmaceutical preparation.

9. The antitumor and / or anti-inflammatory drug according to claim 5, characterized in that, The dosage form of the drug includes any one of capsules, granules, tablets, pills, syrups, powders, granules, suppositories, drops, and emulsions.

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