Ferroptosis-immunotherapy combined therapeutic drug composition and preparation method thereof

Abstract

A ferroptosis-immunotherapy combined therapeutic drug composition and its preparation method, wherein the composition comprises an ssPPE core and an Fe-TA network shell, the ssPPE core is loaded with β-lapachone as an inducer, and the ssPPE core and β-lapachone form a core ssPPE Lap embedded in the Fe-TA network shell; the Fe-TA network shell is a metal polyphenol network obtained by reacting a TA solution and an FeCl3 solution in an alkaline environment; the ssPPE core is a polyphosphate ester containing a disulfide bond synthesized from ethyl dichlorophosphate and 2,2'-dithioglycol. The ssPPE of the present invention Lap @Fe-TA significantly induces ferroptosis of tumor cells through the synergistic effect of increasing Fe 3+ / H2O2 supply and GSH consumption, can effectively inhibit tumor progression. In addition, the amplified ferroptosis induces immunogenic cell death of tumor cells, thereby promoting dendritic cell (DC) maturation and T cell infiltration. Moreover, when combined with anti-PD-L1 antibody (a-PD-L1), ssPPE Lap @Fe-TA significantly inhibits 4T1 tumor growth and lung metastasis.

Description

Technical Field

[0001] The present invention relates to the field of tumor therapeutic drugs, and in particular to a ferroptosis-immunotherapy combined therapeutic drug composition and a preparation method thereof. Background Art

[0002] At present, traditional treatments such as chemotherapy, radiotherapy and surgery are still not ideal for advanced cancer, and the role of ferroptosis in tumor cell death is still unsatisfactory. Previous studies have mainly focused on the development of various iron-based nanomaterials, because very high iron doses are required to produce sufficient intracellular reactive oxygen species (ROS) through the Fenton reaction. In the process of deepening the study of ferroptosis, it was found that phospholipid peroxidase GPX4 can convert cytotoxic LPO into non-toxic ester alcohols by consuming glutathione (GSH) and maintaining the homeostasis of the membrane lipid bilayer, thereby leading to inefficient ferroptosis of tumor cells. In summary, the current induction of ferroptosis has the following problems: 1. Current research mainly focuses on the development of iron-based materials, and the mechanism of intracellular ferroptosis is not so detailed; 2. Fe 3+ The initiation of the mediated Fenton reaction requires a very high dose of iron, which undoubtedly affects biosafety; 3. The essence of ferroptosis is lipid peroxidation. Then the GSH-GPX4 responsible for maintaining redox homeostasis in the cell is what we don’t want. How to remove these becomes the key to solving inefficient ferroptosis and is an important topic of current research. Summary of the invention

[0003] Based on this, the present invention provides a ferroptosis-immunotherapy combined therapy pharmaceutical composition and a preparation method thereof. The pharmaceutical composition increases Fe 3+ / H 2 O 2 On the one hand, ferroptosis is promoted by supplying GSH, and on the other hand, the expression of GPX4 is inhibited by consuming GSH through disulfide bonds. By combining these two pathways, we further amplified ferroptosis, and by combining with anti-PD-L1, we can further enhance tumor treatment.

[0004] To achieve the above object, the present invention provides a ferroptosis-immunotherapy combined therapeutic pharmaceutical composition, which comprises a ssPPE core and a Fe-TA network shell, wherein the ssPPE core is loaded with β-lapachone used as an inducer, and the ssPPE core and β-lapachone form a core ssPPE Lap Embedded in the Fe-TA network shell; the Fe-TA network shell is formed by TA solution and FeCl 3 The metal polyphenol network is obtained by reacting the solution in an alkaline environment; the ssPPE core is a polyphosphate containing a disulfide bond synthesized by ethyl dichlorophosphate and 2,2'-dithiodiethanol.

[0005] As a further preferred technical solution of the present invention, during the preparation process of the ferroptosis-immunotherapy combined therapeutic pharmaceutical composition, the feed mass ratio of the ssPPE core to β-lapachone is 10:1-100:1; the feed mass ratio of the ssPPE core to the Fe-TA network shell is 4:1-4:5. The optimal ratio is 100:75:2, that is, the feed mass ratio of the ssPPE core to β-lapachone is 50:1, and the feed mass ratio of the ss-PPE core to the Fe-TA network shell is 4:3.

[0006] As a further preferred technical solution of the present invention, the pharmaceutical composition is spherical, and the particle size is 70 to 100 nm.

[0007] According to another aspect of the present invention, the present invention also provides a method for preparing a ferroptosis-immunotherapy combined therapeutic pharmaceutical composition, comprising the following steps:

[0008] S1, synthesizing a polyphosphate containing a disulfide bond by combining ethyl dichlorophosphate and 2,2'-dithiodiethanol;

[0009] S2, dispersing the hydrophobic β-lapachone and the disulfide-bonded polyphosphate in an organic solvent, adding them to sterile water, and then ultrasonically emulsifying and concentrating to remove the organic solvent to obtain ssPPE Lap Solution;

[0010] S3. Add a stirrer to the ssPPELap solution and mix the TA solution and FeCl 3 The solutions are added sequentially and repeatedly for multiple times, and then the pH is adjusted to 7.3-7.8 with a NaOH solution to obtain a ferroptosis-immunity combined therapy pharmaceutical composition.

[0011] As a further preferred technical solution of the present invention, in step S2, the mass ratio of β-lapachone to the disulfide bond-containing polyphosphate is 1:50;

[0012] As a further preferred technical solution of the present invention, TA solution and FeCl 3 The mass ratio of the solute in the solution is 4:1.

[0013] As a further preferred technical solution of the present invention, in step S3, TA solution and FeCl 3 The volume ratio of the solution is 1:1, the concentration of TA solution is 40 mg / mL, and the FeCl 3 The concentration of the solution was 10 mg / mL.

[0014] As a further preferred technical solution of the present invention, the organic solvent is chloroform.

[0015] As a further preferred technical solution of the present invention, in step S2, the volume ratio of the organic solvent to the sterile water is 1:10.

[0016] As a further preferred technical solution of the present invention, in step S2, a cell disruptor is used for ultrasonic emulsification, and the ultrasonic emulsification conditions are: power 20%, ultrasonic 5s, stop 2s, and continue for 5min.

[0017] The ferroptosis-immunity combined therapy pharmaceutical composition of the present invention and the preparation method thereof, using the above technical scheme, can achieve the following beneficial effects:

[0018] 1) ssPPE of the present invention Lap The hard shell of @Fe-TA interacts strongly with the cell membrane, enhancing tumor cell uptake; after internalization, the Fe-TA network shell and β-lapachone significantly increase intracellular Fe and H 2 O 2 At the same time, the ssPPE core consumes GSH through a disulfide-thiol exchange reaction, inactivating GPX4. 2 O 2 The synergistic effect of Fe supply and GSH depletion ultimately amplified the ferroptosis of tumor cells and achieved a strong inhibition of tumor growth;

[0019] 2) ssPPE of the present invention Lap @Fe-TA can promote DCs maturation, enhance CD8+ T cell infiltration, proliferation and IFN-γ secretion, and reduce the proportion of immunosuppressive Treg cells, inducing a strong systemic anti-tumor immune response. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0021] Figure 1 (a) Synthesis route and (b) 1H NMR spectrum of disulfide-containing polyphosphate ester (ssPPE);

[0022] Figure 2 (a) Synthesis route and (b) 1H NMR spectrum of polyphosphate ester (ccPPE);

[0023] Figure 3 For ssPPE Lap @Fe-TA and PPE Lap @Fe-TA particle size and potential diagram;

[0024] Figure 4 ssPPE at different feed ratios Lap @Fe-TA particle size;

[0025] Figure 5For ssPPE Lap @Fe-TA and PPE Lap @Fe-TA stability curve;

[0026] Figure 6 For ssPPE Lap @Fe-TA Fe release curve;

[0027] Figure 7 To add ssPPE Lap @Fe-TA or PPE Lap UV-vis spectra of MB aqueous solution after @Fe-TA;

[0028] Figure 8 For PPE Lap @Fe-TA and different concentrations (0.2, 0.5, 1.0 mg mL -1 )ssPPE Lap @GSH consumption capacity after Fe-TA treatment;

[0029] Fig. 9 To use ROSGreen TM H 2 O 2 Probe flow cytometry analysis of intracellular H 2 O 2 concentration;

[0030] Fig.10 For PPE Lap @Fe-TA and different concentrations (0.2, 0.5, 1.0 mg mL -1 )ssPPE Lap @GSH levels in CT26 cells after Fe-TA treatment;

[0031] Fig.11 For ssPPE Lap @The killing effect of Fe-TA on CT26 cells under alternating magnetic field;

[0032] Fig.12 For C11-BODIPY 581 / 591 The probe was used to detect the expression of LPO in CT26 cells after different treatments;

[0033] Fig.13 Based on ssPPE Lap @Schematic diagram of the treatment process of Fe-TA on the CT26 tumor model;

[0034] Fig.14 Based on ssPPE Lap @Tumor growth curve after Fe-TA treatment in CT26 tumor model;

[0035] Fig.15 Based on ssPPE Lap @Image of the tumor removed at the end of Fe-TA treatment in the CT26 tumor model;

[0036] Fig.16 Based on ssPPE Lap @Fe-TA's ferroptosis therapy shows its immunostimulatory effect in vitro;

[0037] Fig.17 Based on ssPPE Lap @Fe-TA's ferroptosis therapy in vivo immunostimulatory effect diagram;

[0038] Fig.18 Based on ssPPE Lap @Schematic diagram of the treatment process of Fe-TA combined with anti-PD-L1 in 4T1 tumor model;

[0039] Fig.19 Based on ssPPE Lap @Tumor growth curve after treatment with Fe-TA combined with anti-PD-L1 in 4T1 tumor model;

[0040] Fig. 20 Based on ssPPE Lap @Image of the tumor removed at the end of treatment with Fe-TA combined with anti-PD-L1 in the 4T1 tumor model;

[0041] Fig.21 Based on ssPPE Lap @Image of lung metastases removed at the end of treatment with Fe-TA combined with anti-PD-L1 in the 4T1 tumor model.

[0042] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings in conjunction with the embodiments. DETAILED DESCRIPTION

[0043] The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0044] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0045] The TA solution in the present invention refers to a tannic acid solution, the full name of which is Tannic acid, abbreviated as TA, and its molecular formula is C76 H 52 O 46 .

[0046] The ferroptosis-immunotherapy drug composition proposed in the present invention can also be called a rigid metal-polyphenol shell modified nanodevice, denoted as ssPPE Lap @Fe-TA, its function is to enhance ferroptosis.

[0047] The ssPPE Lap @Fe-TA consists of a rigid Fe-TA network shell, a ssPPE core of polyphosphate containing disulfide bonds, and β-lapachone (H 2 O 2 The core of ssPPE is loaded with β-lapachone to form a core ssPPE located in the Fe-TA network shell. Lap , Fe-TA network shell coated on the core ssPPE Lap The Fe-TA network shell is formed by TA solution and FeCl 3 The metal polyphenol network is obtained by reacting the solution in an alkaline environment; the ssPPE core is a polyphosphate containing a disulfide bond synthesized by ethyl dichlorophosphate and 2,2'-dithiodiethanol.

[0048] Specifically, the mass ratio of the ssPPE core, Fe-TA network shell, and β-lapachone of the ferroptosis-immunotherapy combined therapeutic pharmaceutical composition is 100:75:2. The pharmaceutical composition is spherical with a particle size of 70 to 100 nm, preferably 85 nm.

[0049] In the drug composition (nanodevice), the hard Fe-TA network shell can be effectively internalized by tumor cells, and then disintegrated in acidic endosomes / lysosomes, activating the Fe 3+ / Fe 2+ Transformation-driven ferroptosis can also lead to entrapment of the core ssPPE Lap Subsequently, the disulfide-containing polyphosphate inactivated GPX4 through a disulfide-thiol exchange reaction, depleting intracellular GSH and triggering the rapid release of β-lapachone, which significantly increased intracellular H 2 O 2 level, further through Fe 3+ The mediated Fenton reaction produces a large amount of ROS, thereby amplifying ferroptosis; ultimately, ssPPE Lap @Fe-TA increases Fe 3+ / H 2 O 2The synergistic effect of GSH supply and depletion significantly induced ferroptosis of tumor cells, thereby effectively inhibiting tumor progression. In addition, amplified ferroptosis induced immunogenic cell death of tumor cells, thereby promoting dendritic cell (DC) maturation and T cell infiltration. When combined with anti-PD-L1 antibody (a-PD-L1), ssPPE Lap @Fe-TA significantly inhibited 4T1 tumor growth and lung metastasis.

[0050] The technical solution of the present invention is described in detail below in conjunction with specific embodiments.

[0051] Example 1

[0052] Step 1, preparation of disulfide bond-containing polyphosphate (ssPPE):

[0053] 2,2'-dithiodiethanol was distilled under reduced pressure, and then the flask containing 2,2'-dithiodiethanol was placed in an oil bath, and the temperature was increased by 10°C every 5 minutes until the temperature of the oil bath reached 160°C. The distillation was observed, the front fraction was discarded, and the fraction with a temperature of 128°C to 130°C was collected;

[0054] Take 75 mL of tetrahydrofuran in a balanced pressure funnel, add 50 mL of tetrahydrofuran (THF) dropwise into the flask, then add the fraction collected at 128°C to 130°C, 16.3 g of ethyl dichlorophosphate (0.1 mol) and 11.1 g of triethylamine (0.11 mol) into the balanced pressure funnel. The fraction contains 15.4 g of 2,2'-dithiodiethanol (0.1 mol), then slowly add it dropwise into the -20°C reaction apparatus through the balanced pressure funnel and react overnight (6 to 12 h).

[0055] The sample that had reacted overnight in a -20°C cold bath was taken out and placed at room temperature for continuous reaction for 48 hours. The experiment was terminated, and the reaction solution was passed through a Buchner funnel to remove diethylamine hydrochloride (white precipitate), and the remaining THF was removed under reduced pressure. The reaction solution was observed to be light brown and gradually viscous, and the above solution was dripped into cold ether at -20°C, stirred for 10 minutes, and then allowed to stand for 1 hour. The precipitation was repeated at least three times, and the ether was removed by rotary evaporation to obtain a product containing a disulfide bond polyphosphate, labeled as ssPPE, and characterized by 1H NMR using DMSO-d6. Figure 1 As shown, it shows that ssPPE was successfully prepared.

[0056] Step 2, ssPPE loading β-lapachone:

[0057] Take 500 μL ssPPE (20 mg / mL, dissolved in CHCl 3) and 200 μL β-lapachone (1 mg / mL, dissolved in CHCl 3 ) in a 50 mL centrifuge tube, fully mixed and dissolved, and then 9 mL of ultrapure water was added; in an ice bath, the product was fully emulsified using an ultrasonic probe for 5 min (20%, on for 5 s, off for 2 s), and then rotary evaporated to remove chloroform and concentrated under certain temperature and pressure conditions to obtain ssPPE Lap The nanoparticles in the obtained solution are labeled as ssPPE Lap .

[0058] Step 3: Fe-TA network shell embedding core ssPPE Lap :

[0059] Then to ssPPE Lap Add a stirring bar to the solution, add 60 μL of TA solution (40 mg / mL, dissolved in water) and 60 μL of FeCl 3 solution (10 mg / mL, dissolved in water), and then added dropwise to the ssPPE Lap The solution was added in three times, and then the pH was adjusted to about 7.5 by dropping NaOH solution. At this time, the liquid was collected and concentrated by centrifugation to obtain a ferroptosis-immunity combined therapy drug composition (nanodevice), which was marked as ssPPE Lap @Fe-TA.

[0060] Comparative Example 1

[0061] This comparative example is a comparative experiment of Example 1, using the same preparation method as Example 1, except that 15-pentanediol (19.5 g, 51.7%) is used to replace 2,2'-dithiodiethanol, and all other conditions remain unchanged. In Comparative Example 1, the product prepared in step 1 is ccPPE, and the product obtained in step 2 is PPE Lap The product obtained in step 3 is PPE Lap @Fe-TA. PPE was characterized by 1H NMR using DMSO-d6. Figure 2 As shown, it shows that the PPE material was successfully prepared.

[0062] Example 2

[0063] This example is a comparative experiment of Example 1, using the same preparation method as Example 1, except that the mass ratio of ssPPE to β-lapachone is different (10:1), and all other conditions remain unchanged. Lap @Fe-TA, particle size Figure 4 shown.

[0064] Example 3

[0065] This example is a comparative experiment of Example 1, using the same preparation method as Example 1, except that the mass ratio of ssPPE to Fe-TA network shell is different (4:5), and all other conditions remain unchanged. Lap @Fe-TA, particle size Figure 4 shown.

[0066] In order to further explore the beneficial technical effects of the present application, the following tests were performed on the product of Example 1, and the product of Comparative Example 1 was used as a control group.

[0067] Test 1: ssPPE Lap @Fe-TA and PPE Lap The characterization of the two @Fe-TA nanodevices is as follows:

[0068] (1) Particle size and potential

[0069] The nanodevices obtained above were tested by dynamic light scattering (DLS) to determine the particle size and potential of the nanodevices. Figure 3 As shown, the particle sizes of the two are basically the same, about 85nm; the electric potentials of the two are also basically the same, about -48mV.

[0070] (2) Particle stability

[0071] like Figure 5 As shown in the figure, the two nanodevices have good stability. After 72 hours of co-culture in BMDM solution, the particle size of the two nanodevices did not change significantly. This is because the Fe-TA metal polyphenol network layer can provide an inert surface for the device particles, thereby improving the stability of the particles.

[0072] (3) ssPPE Lap Drug release behavior of @Fe-TA

[0073] Fe 3+ From ssPPE Lap @Fe-TA release was in the presence of 0.02 mol L -1 The experiment was carried out in phosphate buffered saline (PBS, pH = 7.4 or 5.5). Three parallel experiments were performed, and 1 mL of ssPPE was taken. Lap@Fe-TA ([Fe] = 500 μg / mL) was resuspended in 1 mL 0.02 M PBS (pH = 7.4 or 5.5), and the nanodevice was placed in a dialysis bag (Spectra / Por, Float-A-Lyzer, MWCO = 14000). The dialysis bag was then placed in three sets of 50 mL centrifuge tubes containing 15 mL of PBS buffer (pH = 7.4 or 5.5), and the release was carried out at 37 ° C shaker (80 rpm). At the specified time, the release external liquid was completely removed and supplemented with an equal amount of fresh buffer. 5 mL of the removed release external liquid was collected and marked, and the release external liquid at different time points was uniformly analyzed by ICP-MS. 3+ Quantification of Figure 6 As shown, ssPPE Lap @Fe-TA particles As the pH value decreases, Fe 3+ The release rate and release amount are increased.

[0074] (4) ssPPE Lap Characterization of OH generated by @Fe-TA

[0075] MB (10 μg / mL) and H 2 O 2 (9.8mM), PPE Lap @Fe-TA,ssPPE Lap @Fe-TA and FeCl 3 ([Fe] = 0.5 mM) at 37 ° C to measure the generation of ·OH, and then observe the change in the absorbance of MB at 665 nm. The results are as follows Figure 7 As shown, compared with the negative control group MB solution, MB+H 2 O 2 Compared with solution, ssPPE Lap @Fe-TA and PPE Lap @Fe-TA has a significant MB degradation effect, compared with the positive control group FeCl 3 MB can also be degraded, which shows that Fe 3+ The mediated Fenton reaction can produce ·OH and lead to MB degradation.

[0076] (5) ssPPE Lap Characterization of GSH consumption by @Fe-TA

[0077] Determination of ssPPE using glutathione test kit Lap @Fe-TA's ability to consume glutathione in vitro. PBS, PPE Lap @Fe-TA and different concentrations (0.2 mg / mL, 0.5 mg / mL, 1 mg / mL) of ssPPE LapThe Fe-TA solution and the GSH solution were mixed together and gently shaken (800 rpm) at 37°C. After incubation for 24 hours, the mixture was treated with the relevant reagents of the glutathione kit, and then the GSH content was determined by the absorbance at 412 nm of the UV-visible spectrum. Figure 8 As shown, it was found that PPE Lap @Fe-TA compared to ssPPE Lap @Fe-TA has a strong GSH consumption capacity, and with the increase of concentration, the GSH consumption level becomes higher.

[0078] Test 2: ssPPE Lap @Fe-TA kills tumor cells at the cellular level

[0079] A mouse colorectal cancer cell line (CT26) was selected to investigate the ssPPE Lap @Fe-TA's killing of tumor cells was first performed by ROSGreen TM H 2 O 2 Probe validation of β-lapachone in CT26 cells can upregulate endogenous H 2 O 2 Level, such as Fig. 9 As shown in Figure 2, flow cytometry analysis revealed that after being treated with the β-lapachone-encapsulated nanodevice, the intracellular H 2 O 2 The level of NH4+ increased significantly, which will undoubtedly further amplify the Fenton reaction.

[0080] Previous experiments have verified the function of ssPPE materials to consume GSH at the particle level, but whether it can consume GSH in cells needs further verification. First, CT26 cells were inoculated into a 6-well plate and incubated for 24 hours. After incubation in different nanodevices, the cells were collected and washed multiple times with PBS buffer. The cells were then lysed with 40mL Triton-X-100 lysis buffer, and the precipitate below the lysis buffer was collected by centrifugation. 100mL DTNB solution (400mM) was mixed with 100mL lysis buffer and incubated for 30 minutes. The cellular glutathione content in the mixture was determined by measuring its OD at 412nm. Fig.10 Verified by ssPPE Lap The GSH level of CT26 cells after incubation with @Fe-TA was significantly reduced, which further proved that it was caused by the reaction between ssPPE and GSH.

[0081] CT26 cells were plated at 5 × 10 3The cells were seeded into a 96-well plate at a low density. After incubation for 24 h, the old culture medium was discarded. Different nanodevices were added to the 96-well plate and cultured again at 37 °C for 24 h. The old culture medium was discarded and the culture medium containing MTT (V DMEM :V MTT =5:1, C MTT =5mg / mL), react for more than 4h, carefully remove the supernatant, add 400μL DMSO to each well, shake for 10min to fully dissolve, and measure the OD value of each well at 490nm using an enzyme marker. Fig.11 As shown, ssPPE Lap @Fe-TA has extremely high cytotoxicity. 3+ and β-lapachone concentrations, the viability of CT26 cells was lower; Lap @Fe-TA,ssPPE Lap @Fe-TA+DFO and ssPPE@Fe-TA In comparison, ssPPE Lap @Fe-TA exhibited stronger cytotoxicity, which further verified the synergistic treatment of Fe3+, β-lapachone and ssPPE.

[0082] In order to further confirm that CT26 cell apoptosis is caused by ferroptosis, the production of intracellular LPO was detected. LPO is a marker of ferroptosis, and its expression further indicates the extent of ferroptosis. 581 / 591 -C11 detects intracellular LPO levels, BODIPY 581 / 591 -C11 is a fluorescent LPO detection probe that can be inserted into lipid membranes and oxidized by intracellular LPO. The fluorescence of the oxidation product is green (ex: 485nm, λem: 520nm). In this study, CT26 cells were seeded on each plate with 10 5 After 24 hours of incubation, ssPPE@Fe-TA or PPE Lap @Fe-TA, and various inhibitors (DFO and a-Toc) were used to treat the cells for 4 hours, and then the cells were washed with PBS buffer and incubated in serum-free medium for 20 minutes. Finally, they were repeatedly washed with PBS buffer and confocal images were taken using CLSM. The results are shown in Figure 2. Fig.12 As shown, this indicates that LPO was produced in each group, among which ssPPE Lap @Fe-TA produced the most oxidized LPO, which means the degree of ferroptosis was the strongest, which corresponds to the previous MTT experiment.

[0083] Test 3: ssPPE Lap Study on the efficacy of @Fe-TA in mouse CT26 tumor model

[0084] Mouse colorectal cancer CT26 tumors (10 6 / mouse), seven days later, the tumor-bearing mice were randomly divided into five groups, five mice in each group, and began to receive tail vein administration. The treatment process is shown in the figure. Fig.13 Shown (Ⅰ: PBS, Ⅱ: ssPPE@Fe-TA, Ⅲ: ssPPE Lap @Fe-TA+DFO, IV: PPE Lap @Fe-TA, V:ssPPE Lap @Fe-TA), and the drugs were administered on days 0, 2, 4, and 6. After different treatments, the length and width of the tumors on both ends of the mice were measured with a vernier caliper every two days. The volume of the tumor is (length multiplied by (width squared) divided by 2. The experimental results are shown in Fig.14 and 15 As shown, ssPPE Lap The @Fe-TA group showed the strongest inhibitory effect on tumor growth, which also verified that H 2 O 2 / Fe supply and GSH depletion work synergistically to ultimately amplify ferroptosis of tumor cells.

[0085] Test 4: ssPPE Lap @Fe-TA promotes DC cell maturation in vivo and in vitro

[0086] Colorectal cancer CT26 tumors were implanted on the left side of the back of Balb / c mice. Eleven days later, the mice were randomly divided into five groups, three mice in each group, and the tumors were treated with I: PBS, II: ssPPE@Fe-TA, III: ssPPE Lap @Fe-TA+DFO, IV: PPE Lap @Fe-TA, V:ssPPE Lap @Fe-TA. Three days after administration, the mice were eyeballed and blood was collected, and the inguinal lymph nodes were removed. The inguinal lymph node cells were stained with anti-CD45-BV510, anti-CD11c-PE, anti-CD80-PE / CF594, and anti-CD86-APC flow cytometry antibodies, and the maturation rate of DC in the lymph nodes was tested using a 12-color flow cytometer (BD FACSCelesTA). Fig.16 As shown, ssPPE LapThe maturation rate of DC in the inguinal lymph nodes of the @Fe-TA group was significantly increased. Similarly, mice were killed by cervical dislocation and their femurs were removed. Bone marrow cells in the backbone were collected and inoculated into 24-well plates. The medium was changed every two days (20 ng / mL GM-CSF and 10 ng / mL IL-4 were added to the culture medium). Mouse bone marrow-derived dendritic cells were cultured to the sixth day and could be used. CT26 cells treated with different conditions were (1) PBS (CT26 cells), (2) ssPPE@Fe-TA (ssPPE@Fe-TA+CT26 cells), (3) ssPPE Lap @Fe-TA(ssPPE Lap @Fe-TA+CT26 cells), (4) ssPPE Lap @Fe-TA+DFO(ssPPE Lap @Fe-TA+DFO+CT26 cells). These treated tumor cells were placed in the upper layer of the Transwell system, and the lower layer was DC cells, and incubated for 24 hours. After 24 hours, BMDCs were stained with flow cytometry antibodies. After staining, the maturation rate of DCs was tested using a 12-color flow cytometer (BD FACSCeles TA). Fig.17 As shown, ssPPE Lap @The DC maturation rate in the Fe-TA group was significantly improved.

[0087] Test 5: ssPPE Lap Study on the efficacy of @Fe-TA in the mouse 4T1 lung metastasis model

[0088] Mouse breast cancer tumors (10 6 / mouse), seven days later, the tumor-bearing mice were randomly divided into four groups, five mice in each group, and treated by drug administration through the tail vein. The treatment process is shown in the figure Fig.18 As shown (Ⅰ: PBS, Ⅱ: a-PD-L1, Ⅲ: ssPPE Lap @Fe-TA, IV:ssPPE Lap @Fe-TA+a-PD-L1), ssPPE was administered on days 0, 3, 6, and 9, respectively Lap @Fe-TA, a-PD-L1 was administered on days 1, 4, and 7. After different treatments, the length and width of the tumors on both ends of the mice were measured with a vernier caliper every two days. The volume of the tumor was (length multiplied by (width squared)) divided by 2. The experimental results are shown in Fig.19 and 20 As shown, ssPPE LapThe Fe-TA+a-PD-L1 group showed the strongest effect in inhibiting distal tumor growth, which may be due to the fact that after the synergistic treatment of ferroptosis activated cytotoxic T cells, the use of the immune checkpoint blocker a-PD-L1 further relieved the signal that inhibited T cell activation and maintained T cell activation. Finally, we took the mouse lungs for observation. Fig.21 As shown, we found that ssPPE Lap The number of lung nodules in the mice in the Fe-TA+a-PD-L1 group was very small, which was significantly different from that in the PBS group, indicating that ssPPE Lap @Fe-TA+a-PD-L1 also has significant therapeutic effect in inhibiting lung metastasis.

[0089] Although specific embodiments of the present invention are described above, those skilled in the art should understand that these are merely examples and that various changes or modifications may be made to the embodiments without departing from the principles and essence of the present invention. The scope of protection of the present invention is limited only by the appended claims.

Claims

1. A ferroptosis-immunotherapy combined pharmaceutical composition, characterized in that: The invention comprises a ssPPE core and a Fe-TA network shell, wherein the ssPPE core is loaded with β-lapachone as an inducer, and the ssPPE core and β-lapachone form a core ssPPE Lap Embedded in the Fe-TA network shell; the Fe-TA network shell is a metal polyphenol network obtained by reacting TA solution and FeCl3 solution in an alkaline environment; the ssPPE core is a disulfide-bonded polyphosphate synthesized by ethyl dichlorophosphate and 2,2'-dithiodiethanol; wherein, During the preparation of the ferroptosis-immunotherapy combined therapeutic drug composition, the mass ratio of the ssPPE core to β-lapachone is 10:1-100:1; the mass ratio of the ssPPE core to the Fe-TA network shell is 4:1-4:5; The TA solution refers to a tannic acid solution.

2. The ferroptosis-immunotherapy combined therapeutic pharmaceutical composition according to claim 1, characterized in that: The pharmaceutical composition is spherical and has a particle size of 70-100 nm.

3. A method for preparing the ferroptosis-immunotherapy combined therapeutic pharmaceutical composition according to claim 1 or 2, characterized in that: The following steps are involved: S1, synthesizing a polyphosphate containing a disulfide bond by combining ethyl dichlorophosphate and 2,2'-dithiodiethanol; S2, dispersing the hydrophobic β-lapachone and the disulfide-bonded polyphosphate in an organic solvent, adding them to sterile water, and then ultrasonically emulsifying and concentrating to remove the organic solvent to obtain ssPPE Lap Solution; S3, in ssPPE Lap A stirring bar is added to the solution, and the TA solution and the FeCl3 solution are added sequentially and repeatedly for multiple times, and then the pH is adjusted to 7.3-7.8 with a NaOH solution to obtain a ferroptosis-immunotherapy combined therapeutic pharmaceutical composition.

4. The method for preparing the ferroptosis-immunotherapy combined therapeutic pharmaceutical composition according to claim 3, characterized in that: In step S2, the mass ratio of β-lapachone to the disulfide bond-containing polyphosphate is 1:

50.

5. The method for preparing the ferroptosis-immunotherapy combined therapeutic pharmaceutical composition according to claim 3, characterized in that: In step S3, the mass ratio of the solute in the TA solution to that in the FeCl3 solution is 4:

1.

6. The method for preparing the ferroptosis-immunotherapy combined therapeutic pharmaceutical composition according to claim 5, characterized in that: In step S3, the volume ratio of TA solution to FeCl3 solution is 1:1, the concentration of TA solution is 40 mg / mL, and the concentration of FeCl3 solution is 10 mg / mL.

7. The method for preparing the ferroptosis-immunotherapy combined therapeutic pharmaceutical composition according to claim 3, characterized in that: The organic solvent is chloroform.

8. The method for preparing the ferroptosis-immunotherapy combined therapeutic pharmaceutical composition according to claim 3, characterized in that: In step S2, the volume ratio of the organic solvent to sterile water is 1:

10.

9. The method for preparing the ferroptosis-immunotherapy combined therapeutic pharmaceutical composition according to claim 3, characterized in that: In step S2, a cell disruptor is used for ultrasonic emulsification, and the ultrasonic emulsification conditions are: power 20%, ultrasonic 5 s, stop 2 s, and continue for 5 min.

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