Iron polyphenol nanocapsule, and preparation method and application thereof

Abstract

This invention relates to an iron polyphenol nanocapsule, its preparation method, and its applications. The nanocapsule is prepared from raw materials containing ferric salts, ferric ions, and gossypol via a microfluidic chip. The iron polyphenol nanocapsule containing gossypol-ferric ions-ferric ions prepared by this invention using a microfluidic chip as a reactor exhibits uniform size and good stability. It can achieve co-delivery of dual chemotherapy drugs, T1 magnetic resonance imaging, and trimodal combined therapy of tumors (chemodynamic therapy / dual-channel chemotherapy / immunotherapy), showing potential applications in cancer diagnosis and treatment.

Description

Technical Field

[0001] This invention belongs to the field of functional nanomaterials, and specifically relates to an iron polyphenol nanocapsule, its preparation method, and its application. Background Technology

[0002] Cancer remains one of the leading causes of death worldwide. Traditional treatments such as surgery, radiotherapy, and chemotherapy suffer from unsatisfactory efficacy, significant side effects, and a high risk of recurrence and metastasis. Chemotherapy, with its advantages of universality, non-invasiveness, and systemic anti-tumor effects, remains a primary treatment for cancer in clinical practice. Chemotherapy inhibits cancer cell proliferation and prevents cancer cell invasion and metastasis, thus achieving the goal of treating cancer. However, chemotherapy alone is insufficient to address the complexity and heterogeneity of tumors, often leading to unsatisfactory treatment outcomes, drug resistance, and severe adverse reactions. Furthermore, chemotherapy drugs often suffer from low bioavailability, short blood circulation time, poor targeting, and strong toxic side effects, resulting in unsatisfactory clinical efficacy and prognosis. Currently, methods to improve the effectiveness of chemotherapy mainly include two aspects. One is the use of nanocarriers to load chemotherapy drugs, prolonging their blood circulation time and increasing their accumulation at the tumor site, thereby achieving the goal of "reducing toxicity and increasing efficacy." Various nanocarriers have already been used in research on the delivery of chemotherapy drugs. However, the in vivo toxicity of the nanocarrier's own components, limited drug loading capacity, and cost-effectiveness often restrict its further clinical translation. Therefore, developing low-toxicity or even non-toxic nanocarriers with simple components and assembling nanoparticles directly from the drug itself without destroying drug activity—that is, "carrier as drug"—is of great significance for promoting the clinical translation of nanomedicines. On the other hand, synergistic therapies mediated by two or more drugs and the combination of multiple treatment modalities can significantly improve the efficacy of cancer treatment. Therefore, developing multifunctional nanomedicine delivery platforms with simple components is crucial for improving tumor treatment and reducing systemic toxicity.

[0003] Tumor imaging provides a basis for cancer diagnosis and enables real-time monitoring of treatment efficacy and the in vivo metabolism of nanomedicines. Iron-based nanoplatforms, possessing both chemokinetic therapy (CDT) and magnetic resonance (MR) imaging properties, are considered potential therapeutic nanoplatforms, such as iron oxide, metal-organic iron frameworks (MOF(Fe)), ferrous disulfide (FeS2), and iron-based metal network nanoparticles. Due to their simple structure and composition, ease of synthesis, and multifunctionality, iron-based metal polyphenol networks have attracted widespread attention in the development of nanomedicines for cancer diagnosis and treatment. 3+ On the one hand, it can consume glutathione (GSH) and reduce it to Fe. 2+ On the other hand, it can be achieved through Fe 3+ Fenton-like mediated reaction and Fe2 + The Fenton-mediated reaction, together producing ROS, achieves CDT in cancer cells, inducing oxidative stress. Interestingly, Fe, formed through redox reactions... 3+ and Fe 2+ The classical cyclical reactions between these components can continuously regulate the tumor microenvironment (TME) and reduce the antioxidant capacity of cancer cells. Therefore, developing multifunctional iron-based nanoplatforms for MR imaging and CDT-mediated combined therapy of tumors is of great significance.

[0004] A search of domestic and international literature and patents revealed that no method has been reported for preparing gossypol-ferric ion-Fungamicin iron polyphenol nanocapsules based on microfluidic chips for the combined T1 MRI-guided chemodynamic therapy / dual-channel chemotherapy / immunotherapy trimodal treatment of tumors. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an iron polyphenol nanocapsule, its preparation method and application, to overcome the defects of single tumor treatment, improve the enrichment of chemotherapy drugs at the tumor site, enhance the anti-tumor effect and reduce its side effects.

[0006] The present invention discloses an iron polyphenol nanocapsule, wherein the nanocapsule is obtained from raw materials containing ferric salt, fentanylmycin, and gossypol via a microfluidic chip.

[0007] The nanocapsules are obtained by a one-step reaction using gossypol, ferric salts, and fungamycin as raw materials, with the flow ratio controlled.

[0008] The nanocapsules are gossypol-ferric ion-Fungamicin iron polyphenol nanocapsules.

[0009] The microfluidic chip includes five inlets, one outlet, and an S-shaped microfluidic channel.

[0010] The present invention provides a method for preparing iron polyphenol nanocapsules, comprising:

[0011] (1) Mix ferric salt, gossypol, water and ethanol, and sonicate to obtain a mixed solution of gossypol-ferric intermediate;

[0012] (2) Mix ferric salt, fugamycin, water and ethanol, and sonicate to obtain a mixed solution of fugamycin iron intermediate;

[0013] (3) Using a microfluidic chip, the mixed solution of fulvamycin iron intermediate was injected into the first injection port, the mixed solution of gossypol iron intermediate was injected into the second injection port, and the buffer solution was injected into the third and fourth injection ports respectively. The iron polyphenol nanocapsules were collected through the outlet.

[0014] The preferred embodiment of the above preparation method is as follows:

[0015] In step (1), the ferric salt is anhydrous ferric chloride; the ratio of the ferric salt, gossypol, water, and ethanol is 0.08-0.15 mg: 0.85-1.25 mg: 10-30 μL: 120.5-145.5 μL; and the ultrasonic time is 1-5 min.

[0016] In step (2), the ferric salt is anhydrous ferric chloride; the ratio of the ferric salt, fulvamycin, water, and ethanol is 0.20-0.40 mg: 0.45-0.65 mg: 3.55-5.55 mL: 0.54-0.75 mL; and the ultrasonic time is 1-5 min.

[0017] In step (3), the buffer solution is Tris buffer with a pH of 7.4–10.5; the ratio of gossypol, ferric salt (the total concentration of gossypol in the reaction system is 0.15–0.35 mg / mL, and the total concentration of ferric salt is 0.05–0.10 mg / mL), and fulvamycin is 0.15–0.35 mg / mL: 0.05–0.10 mg / mL: 0.08–0.20 mg / mL.

[0018] In step (3), the flow rate ratio of the first injection port, the second injection port, the third injection port and the fourth injection port is 3~6:0.1~0.3:35~55:3~6.

[0019] Furthermore, the flow rate ratio of the first injection port, the second injection port, the third injection port and the fourth injection port is 3-6 mL / h: 0.1-0.3 mL / h: 35-55 mL / h: 3-6 mL / h.

[0020] Preferably, the flow rate ratio of the first injection port, the second injection port, the third injection port and the fourth injection port is 4.8 mL / h: 0.16 mL / h: 49.6 mL / h: 4.8 mL / h.

[0021] The microfluidic chip in step (3) includes: a first inlet, a second inlet, a third inlet, a fourth inlet, an S-shaped microfluidic channel, and an outlet connected in sequence; wherein the first inlet, the second inlet, the third inlet, the fourth inlet, the S-shaped microfluidic channel, and the outlet are connected through the microfluidic channel, and the third inlet includes a third sub-inlet 1 and a third sub-inlet 2, which are located on both sides of the microfluidic channel.

[0022] The reaction solution volume and flow rate of the two sub-injections of the third injection port are the same.

[0023] The height of each microfluidic channel is 30–100 μm, the width of the microfluidic channels of the first, second, and third inlets is 80–150 μm, the width of the remaining microfluidic channels is 200–400 μm, and the total length from the channel inlet to the outlet is 30.5–38.5 mm.

[0024] In step (3), the sample is added simultaneously. The corresponding volume of reaction solution is drawn with a syringe and then fixed on the corresponding micro-injection pump. After all the reaction solutions are prepared, the parameters of the micro-injection pump are set. After the receiving device is prepared, the start button of all micro-injection pumps is clicked to start the reaction.

[0025] The method for fabricating the microfluidic chip includes: designing an S-shaped microfluidic channel structure containing five inlets and one outlet, then printing a mask, then photolithographically fabricating a mold on a silicon wafer, and then casting to obtain a microfluidic channel cover sheet; using a glass slide as a substrate and the microfluidic channel cover sheet, and then plasma bonding them to obtain the microfluidic chip.

[0026] The plasma bonding process parameters are: vacuum degree of 18-22 Pa and bonding treatment time in air of 70-90 s.

[0027] The present invention relates to the application of the iron polyphenol nanocapsules in the preparation of a trimodal antitumor drug for chemodynamic therapy / dual-channel chemotherapy / immunotherapy or a multimodal inhibitory drug for T1 magnetic resonance imaging and enhanced tumor cell proliferation.

[0028] The present invention relates to the application of the iron polyphenol nanocapsule combined with PD-L1 antibody (A-PD-L1) in the preparation of an antitumor drug.

[0029] This invention synthesizes gossypol-ferric ion-fungamycin iron polyphenol nanocapsules (GFT NCAs) using a microfluidic chip with a mixture of gossypol and ferric chloride, a mixture of fungamycin and ferric chloride, and Tris buffer (pH=10.5). Gossypol can cause mitochondrial dysfunction, activate the mitochondrial apoptosis pathway, and lead to cancer cell apoptosis. Fungamycin blocks the IRE1α-XBP1 signaling pathway, exacerbates ERS, and prevents cancer cells from restoring homeostasis, thereby inducing cancer cell apoptosis. Ferric ions coordinate with the polyphenol structures of gossypol and fungamycin to form iron polyphenol nanocapsules. Ferric ions can consume excess GSH in the TME, disrupt the redox balance in cancer cells, and reduce the antioxidant capacity of cancer cells. On the other hand, they can also react with hydrogen peroxide (H2O2) to produce ROS, which is used for CDT. At the same time, the cycling between ferric and ferrous ions inside cancer cells allows the Fenton reaction to continue, continuously consuming intracellular GSH and producing ROS, and continuously regulating the TME. Based on the good r1 relaxation rate of ferric ions and the combination therapy-mediated immunogenic death (ICD) of cancer cells, A-PD-L1-mediated immune checkpoint blockade can be further combined in the process of tumor treatment to achieve T1 MRI-guided chemodynamic therapy / dual-channel chemotherapy / immunotherapy trimodal TME-responsive tumor therapy.

[0030] This invention characterizes the physical and chemical properties of prepared gossypol-ferric ion-fungamicin iron polyphenol nanocapsules (GFT NCAs) using ultraviolet-visible absorption spectroscopy (UV-Vis), Zeta potential and dynamic light scattering analysis (DLS), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and inductively coupled plasma optical emission spectroscopy (ICP-OES). The T1MRI performance of GFT NCAs was determined using nuclear magnetic resonance imaging (NMR). The cytotoxicity of GFT NCAs was evaluated using the CCK-8 assay. Laser confocal microscopy (CLSM) was used to verify that GFT NCAs induced mitochondrial membrane dysfunction, exacerbated endoplasmic reticulum stress, and induced immunogenic cell death (ICD) in cancer cells. Flow cytometry was used to detect the effects of GFT NCAs on cancer cell apoptosis and dendritic cell maturation. Finally, a 4T1 mouse subcutaneous breast tumor model was constructed to investigate the T1MRI efficacy of GFT NCAs and the combined treatment effect of chemodynamic therapy / dual-channel chemotherapy / immunotherapy. (See attached product manual.) Figure 1 This diagram illustrates the synthesis and application of the gossypol-ferric ion-fungamicin iron polyphenol nanocapsules (GFT NCAs) based on a microfluidic chip prepared in this invention. Specific test results are as follows:

[0031] 1. Results of Zeta potential and hydrodynamic diameter measurements

[0032] See the instruction manual appendix Figure 2 The hydrodynamic diameter and potential of gossypol-ferric ion-fumigin iron polyphenol nanocapsules (GFT NCAs) are shown. The potential of GFT NCAs is -22.3 ± 0.5 mV, and the hydrated particle size is 185.5 ± 2.9 nm, indicating that GFT NCAs possess ideal size and surface potential. (See attached image.) Figure 3 As shown, the hydrodynamic diameter of GFT NCAs in PBS solution remains unchanged for a relatively long time, demonstrating that GFT NCAs have good colloidal stability.

[0033] 2. Ultraviolet (UV-Vis) Test Results

[0034] See the instruction manual appendix Figure 4 A shows the UV-Vis absorption spectra of Gos, Toy, and GFT NCAs, respectively. The absorption peaks at 234 nm and 374 nm are characteristic peaks of Gos, and the absorption peak at 279 nm is a characteristic peak of Toy. GFT NCAs exhibit a new absorption peak at 251 nm, and the characteristic peak of Gos at 374 nm in GFT NCAs redshifts to 383 nm. These changes are attributed to the interaction of Gos and Fe. 3+ The coordination between Gos-Fe and Toy indicates the formation of Gos-Fe 3+ -Toy's metal polyphenol network structure. Furthermore, GFT NCAs exhibit a new absorption band in the 500-800 nm wavelength range, attributed to Gos-Fe. 3+ The aggregated structure of -Toy demonstrates the successful preparation of GFT NCAs.

[0035] 3. FTIR test results

[0036] See the instruction manual appendix Figure 4 B, infrared spectra of Gos, Toy, and GFT NCAs, respectively, 597 cm⁻¹. -1 The absorption peak at 2961 cm⁻¹ is a characteristic peak of the Fe-O bond in GFT NCAs; -1 and 1380cm -1 These are the characteristic peaks of the CH bond stretching vibration and bending vibration of the methyl group in Gos, respectively, at 1455 cm⁻¹. -1 This is the characteristic peak of the C=C stretching vibration of the benzene ring in Gos; 1025 cm⁻¹ -1 The peak represents the stretching vibration characteristic of the CN bond in Toy. This indicates the presence of the Gos, Toy, and Fe-O coordination structure in GFT NCAs, further proving the successful preparation of GFT NCAs.

[0037] 4. X-ray photoelectron spectroscopy (XPS) test results

[0038] See the instruction manual appendix Figure 5 The images shown are XPS and high-resolution Fe 2p XPS spectra of GFT NCAs, respectively, as attached. Figure 5 As shown in Figure A, four elements appeared in the XPS spectra of GFT NCAs, including Fe, O, C, and N. The characteristic peak of Fe originated from Fe. 3+ The characteristic peaks of N originate from Toy, while the characteristic peaks of O and C are attributed to Gos and Toy. High-resolution XPS spectra of Fe 2p in GFT NCAs (attached) Figure 5 B) can be fitted to four photoelectron peaks at 711.69, 725.51, 717.54, and 732.42 eV, corresponding to Fe 2p 3 / 2 Fe 2p 1 / 2 Fe 2p 3 / 2 satellite and Fe 2p 1 / 2 The binding energy of the satellite proves the Fe in GFT NCAs. 3+ The existence of.

[0039] 5. TEM Test Results

[0040] See the instruction manual appendix Figure 6 This is a TEM image of GFT NCAs, which are regular nanocapsules with uniform size.

[0041] The average particle size is 101.9 ± 7.4 nm.

[0042] 6. Test results of material T1 relaxation properties

[0043] See the instruction manual appendix Figure 7 Figures A and B show the changes in T1 relaxation performance of GFT NCAs under different conditions and their T1 MRI images, respectively. Under neutral conditions, the r1 relaxation rate of GFT NCAs is 2.76 mM. -1 s -1 Furthermore, its T1 MR imaging signal increased with increasing iron concentration, indicating good T1 MR imaging performance. Notably, the r1 of GFT NCAs increased to 3.71 mM under pH 6.5 conditions. -1 s -1 The value of r1 was significantly higher than that at pH 7.4, indicating that the prepared GFT NCAs have the potential to enhance T1 MR imaging in acidic TME. This is mainly attributed to the acid-responsive release of Fe by the GFT NCAs. 3+ The increased content and improved water proton and Fe content 3+The interaction between them. This indicates that, with a microacid response mechanism, GFT NCAs have enhanced T1MR relaxation properties and can serve as good T1MRI contrast agents.

[0044] 7. Results of in vitro ROS generation test

[0045] See the instruction manual appendix Figure 8 A. By investigating the degradation of methylene blue (MB) under different conditions, the generation of reactive oxygen species (ROS) by GFTNCAs under corresponding conditions was examined. It was found that almost no ROS was generated in the absence of GFTNCAs. In a neutral environment containing GFTNCAs, a small amount of MB degradation (20.7%) was clearly observed, which is due to the small amount of Fe released by GFTNCAs. 3+ It undergoes a Fenton-like reaction with H₂O₂, producing a small amount of ROS. Under weakly acidic conditions, the degradation rate of MB significantly increased (52.6%), which is attributed to the weakly acidic environment promoting the dissociation of GFT NCAs, leading to a large amount of Fe. 3+ The release of [a substance] leads to a Fenton-like reaction with H2O2, generating a large amount of ROS, which causes the degradation of MB. (See attached image.) Figure 8 B shows the degradation of MB at different time points when GFTNCAs and H2O2 coexist in a weakly acidic environment at pH 6.5. The insets in the upper left corner show the degradation of samples at 0 and 110 min, respectively. The figures show that MB is gradually degraded by the ROS generated during the reaction over time.

[0046] 8. Drug release test results

[0047] See the instruction manual appendix Figure 9A, 9B, and 9C represent the release curves of Gos, Fe, and Toy from GFT NCAs under different conditions (pH = 7.4, pH = 6.5), respectively. The release amounts of Gos, Fe, and Toy from GFT NCAs over time under different pH conditions were detected using a UV-Vis spectrophotometer and ICP-OES, and the cumulative release rates were calculated based on the initial Gos, Fe, and Toy contents. It can be seen that under weakly acidic conditions (pH = 6.5), after 96 hours, the cumulative release amounts of Gos, Fe, and Toy from GFT NCAs reached 16.9%, 14.2%, and 62.9%, respectively, which are 3.0, 2.2, and 3.3 times higher than the cumulative release amounts of Gos (5.7%), Fe (6.4%), and Toy (19.0%) under physiological conditions (pH 7.4), respectively. This indicates that GFT NCAs can responsively release Gos, Fe, and Toy under acidic TME conditions to achieve highly efficient and specific tumor therapy. Under physiological conditions, GFT NCAs exhibit lower drug release, thereby reducing toxic side effects on normal tissues.

[0048] 9. Cytotoxicity test results

[0049] See the instruction manual appendix Figure 10 A and 10B are CCK-8 cell viability assays of 4T1 cells under different treatments. (See attached image.) Figure 10 As shown in Figure A, 4T1 cells treated with different methods all exhibited a decrease in cell viability dependent on either [Gos] or [Toy], and the half-inhibitory concentrations (IC50) of Gos+Toy (GT) and GFT NCAs on 4T1 cells were calculated. 50 The concentrations (s) were 2.8 and 4.4 μg·mL, respectively. -1 ([Toy]), or 10.1 and 18.1 μg·mL, respectively. -1 ([Gos]), where the individual Toy and Gos ICs 50 s were 3.4 μg·mL -1 ([Toy]) and 15.6 μg·mL -1 ([Gos]). Clearly, GT([Toy] = 2.48 μg·mL -1 Or [Gos] = 10.2 μg·mL -1 GT showed stronger cytotoxicity than Gos or Toy alone, demonstrating the superior anticancer activity of dual-pathway chemotherapy. At this concentration, GT exhibited higher cytotoxicity than GFT NCAs, which may be related to the release of Gos, Toy, and Fe from GFT NCAs in 4T1 cells. 3+ The quantity is related. (See attached document.) Figure 10 As shown in Figure B, 4T1 cells treated differently also exhibited [Fe 3+[Dependent decrease in cell viability, however, Fe alone] 3+ Its anticancer activity is limited; further calculations yielded Gos+Fe 3+ (GF), Toy+Fe 3+ (TF) and GFT NCAs on IC50 in 4T1 cells 50 The values ​​of s were 13.7, 12.5, and 17.5 μg·mL, respectively. -1 ([Fe 3+ It can be observed that by jointly introducing Toy and Fe... 3+ In combination chemotherapy / CDT therapy, TF showed higher cytotoxicity than GF, which may be due to the stronger cytotoxicity of TF and the effect of chemotherapy combined with CDT. When [Fe 3+ = 20.0 μg·mL -1 At this concentration, TF showed stronger cytotoxicity than GFT NCAs, primarily attributed to the drug release efficiency of GFT NCAs in 4T1 cells. At this concentration, based on combination therapy with chemotherapy and CDT, GFT NCAs exhibited better cytotoxicity than Fe. 3+ Much higher cytotoxicity. Overall, GFTNCAs exhibit good antitumor activity.

[0050] 10. Cellular uptake test results

[0051] See the instruction manual appendix Figure 11 Results of cellular uptake assay. GFT NCAs and free Fe 3+ Both exhibited time-dependent cellular uptake. Compared to free Fe... 3+ After co-incubation for 4 hours, the amount of GFT NCAs taken up by cells was significantly higher, indicating that GFT NCAs with nanoscale size are more conducive to the phagocytosis of cancer cells.

[0052] 11. Blood compatibility test results

[0053] See the instruction manual appendix Figure 12 The results are for blood compatibility testing. Water was used as the positive control, PBS buffer as the negative control, and different concentrations of GFT NCAs were used as experimental groups. Each material was co-incubated with Balb / c mouse erythrocyte suspension for 2 hours. UV-Vis absorbance analysis revealed that the hemolysis rate in all GFT NCA groups was less than the threshold of 5%, while the hemolysis rate in the positive control group was significantly higher, indicating that GFT NCAs have good blood compatibility.

[0054] 12. Results of intracellular ROS content test

[0055] See the instruction manual appendix Figure 13A represents the detection of intracellular ROS levels by flow cytometry. PBS was used as the blank control group, and Toy, Gos, and Fe were used as the control groups. 3+ GT, TF, GF, or GFT NCAs ([Fe] = 20 μg / mL, [Gos] = 20.4 μg / mL, and [Toy] = 4.96 μg / mL) were used as experimental groups. After co-incubating with 4T1 cells in an incubator for 6 h, the cells were washed three times with PBS. Under light-protected conditions, 2 μL of ROS probe and 2000 μL of DMEM medium were added to each well, and the cells were incubated for 20 min. After incubation, the cells were washed three times with PBS, then digested with trypsin, centrifuged at 1000 rpm for 5 min to collect the cells, resuspended in PBS, and intracellular ROS content was detected by flow cytometry. (See attached image) Figure 13 As shown in Figure A, compared to the PBS control group, the use of Toy, Gos, or Fe... 3+ ROS levels in the treated cells were significantly improved. Toy amplified the endoplasmic reticulum stress effect in cancer cells, thereby affecting intracellular ROS levels. Similarly, Gos increased intracellular ROS levels, decreased mitochondrial membrane potential, caused mitochondrial dysfunction, and induced oxidative stress in cancer cells. 3+ On one hand, it undergoes a Fenton-like reaction with intracellular H2O2, thereby producing ROS and Fe. 2+ Fe 2+ The Fenton-mediated response further increased intracellular ROS levels. Furthermore, the intracellular ROS levels in the combination therapy groups (GT, TF, and GF) were significantly higher than those in the single therapy groups (Toy, Gos, and Fe). 3+ This indicates that combined therapy is more conducive to the generation of intracellular ROS. Of course, GFT NCAs exhibited the strongest ROS generation capacity across all groups, primarily due to Toy, Gos, and Fe... 3+ The three have a synergistic promoting effect on intracellular ROS generation.

[0056] 13. Results of intracellular GSH content assay

[0057] See the instruction manual appendix Figure 13 B represents the result of intracellular GSH content testing. PBS was used as the blank control group, and Toy, Gos, and Fe were used as the control groups. 3+GT, TF, GF, or GFT NCAs ([Fe] = 20 μg / mL, [Gos] = 20.4 μg / mL, and [Toy] = 4.96 μg / mL) were used as experimental groups. After co-incubating with 4T1 cells in an incubator for 4 h, the cells were washed three times with PBS, digested with trypsin, centrifuged at 1000 rpm for 5 min, resuspended in 500 μL PBS, centrifuged again to collect the cell pellet, and the intracellular GSH level was detected using a GSH detection kit. (See attached...) Figure 13 As shown in Figure B, compared to the PBS group, the other groups all showed varying degrees of GSH depletion, corresponding to the ROS generation capacity mentioned earlier. Among them, Fe... 3+ It exhibits good GSH consumption capacity, which is due to Fe 3+ It can be reduced to Fe by high concentrations of GSH. 2+ And Fe 2+ The ROS generated by the Fenton-mediated reaction can further consume GSH and regenerate Fe. 3+ Interestingly, Fe 3+ and Fe 2+ The classic cyclical response between these factors can lead to sustained GSH depletion and ROS generation, thereby modulating the TME. Similarly, ROS generation mediated by combination therapies (GT, TF, and GF) also exhibits significantly higher GSH depletion capacity than the single-treatment groups (Toy, Gos, and Fe). 3+ Furthermore, the GFT group exhibited the highest GSH consumption, with an intracellular GSH content of only 20.23%. This was attributed to the promoting effect of the combination therapy on intracellular ROS generation and the presence of Fe... 3+ and Fe 2+ The classic cyclic reaction between them mediates continuous TME regulation. In summary, GFT NCAs possess excellent TME regulation capabilities.

[0058] 14. Results of intracellular LPO content assay

[0059] See the instruction manual appendix Figure 14 This is the result of an intracellular LPO content assay. PBS was used as the blank control group, and Toy, Gos, and Fe were used as the assay results. 3+ GT, TF, GF, and GFT NCAs were used as experimental groups. After co-incubating with 4T1 cells in an incubator for 6 hours, the cells were washed three times with PBS. Under light-protected conditions, 1 μL of LPO probe and 500 μL of LMEM medium were added to each well, and the cells were incubated for 20 minutes. After incubation, the cells were washed three times with PBS, fixed with 2.5% glutaraldehyde for 15 minutes, stained with DAPI for 5 minutes, and then the red, green, and blue fluorescence signals of the cells were observed under an oil immersion microscope. (See attached image.) Figure 14As shown, compared with the PBS control group, all other groups exhibited enhanced green fluorescence signal (oxidized C11-BODIPY). 581 / 591 ) and the correspondingly weakened red fluorescence signal (non-oxidized C11-BODIPY) 581 / 591 ), indicating Toy, Gos, Fe 3+ GT, TF, GF, and GFT NCAs can all induce LPO accumulation in cancer cells. Compared with the PBS, Toy, and Gos groups, Fe... 3+ The red fluorescence signal of the treated cells was significantly reduced, while the green fluorescence signal was significantly enhanced. This is attributed to Fe. 3+ Mediated ROS generation and GSH consumption. And compared with single treatment groups (Toy, Gos, and Fe) 3+ Compared to the GT, TF, and GF groups, the cells showed a further decrease in red fluorescence intensity and a corresponding increase in green fluorescence intensity, indicating that the combined treatment significantly promoted intracellular LPO accumulation. Furthermore, based on the combined treatment-mediated ROS generation and GSH consumption, the LPO accumulation effect was most significant in the GFT group.

[0060] 15. Results of mitochondrial membrane potential assay

[0061] See the instruction manual appendix Figure 15 These are the results of a mitochondrial membrane potential assay. PBS was used as the blank control group, and Fe... 3+ Toy, Gos, TF, GT, GF, and GFT NCAs were used as experimental groups. After co-incubating with 4T1 cells in an incubator for 6 hours, the cells were washed three times with PBS. Under light-protected conditions, 1 mL of JC-1 staining working solution and 1 mL of fresh culture medium were slowly added to each well, mixed, and co-cultured for 20 min. The supernatant of old culture medium was removed, and the cells were gently washed twice with JC-1 staining buffer. 2 mL of DMEM culture medium was added to each well, and then the red and green fluorescence signals of the cells were observed using CLSM. (See attached...) Figure 15 As shown, compared with the PBS control group, Fe 3+ The mitochondria of cancer cells in the Toy and Gos groups showed green fluorescence, indicating that Fe... 3+ Toy and Gos can all reduce mitochondrial membrane potential to varying degrees, possibly due to Fe... 3+ The effects of Toy-mediated ROS generation, Toy-amplified endoplasmic reticulum stress on mitochondrial membrane potential, and Gos-mediated mitochondrial dysfunction. Compared to the single-treatment group (Fe... 3+ The combination therapy group (including Toy and Gos) showed stronger green fluorescence and correspondingly weaker red fluorescence. The GFT group exhibited the weakest red fluorescence and the strongest green fluorescence, indicating that GFT NCAs can induce significant mitochondrial dysfunction and help induce apoptosis in cancer cells.

[0062] 16. Results of endoplasmic reticulum stress test

[0063] See the instruction manual appendix Figure 16 This represents the results of an endoplasmic reticulum stress assay. PBS was used as the blank control group, and Fe... 3+ Gos, Toy, GF, TF, GT, and GFT NCAs were used as experimental groups and co-incubated with 4T1 cells in an incubator for 24 h. The culture medium was discarded, and the cells were slowly washed three times with PBS, followed by trypsin digestion to collect the cells. Cells were then lysed and proteins were extracted. Protein concentration was determined, and proteins were separated by SDS-PAGE electrophoresis. Cells were then sequentially transferred, blocked, and incubated with primary and secondary antibodies. Imaging was performed using a chemiluminescence / fluorescent gel imaging system, and finally, protein content was quantified using ImageJ. (See attached image.) Figure 16 As shown, after treatment with different materials, the expression levels of the endoplasmic reticulum stress marker protein GRP78 and the p-IRE1α protein related to the endoplasmic reticulum stress homeostasis recovery pathway p-IRE1α-XBP1 were upregulated to varying degrees. This is due to Fe 3+ Gos can disrupt the redox balance in cancer cells, inducing oxidative stress and thus exacerbating endoplasmic reticulum (ER) stress. Toy, on the other hand, inhibits the restoration of ER homeostasis by blocking the p-IRE1α-XBP1 pathway, thereby inducing increased ER stress. Interestingly, both Toy and Toy-related groups showed upregulation of XBP1u protein expression and downregulation of the corresponding XBP1s protein expression. This is attributed to Toy blocking the p-IRE1α-XBP1 pathway, preventing p-IRE1α from cleaving XBP1u, thus reducing XBP1s expression and leading to further upregulation of XBP1u expression. Moreover, GFT NCAs induced the most significant upregulation of GRP78, p-IRE1α, and XBP1u, and the most significant downregulation of XBP1s, indicating that GFT NCAs have the ability to significantly exacerbate ER stress in cancer cells. Based on this, the expression levels of CHOP, a marker protein of apoptosis induced by ER stress pathways in cancer cells under different treatments, were further investigated. Cancer cells treated with different methods all showed varying degrees of CHOP upregulation, with GFT NCAs inducing the most significant CHOP upregulation, indicating that GFT NCAs can significantly induce cancer cell apoptosis through the endoplasmic reticulum stress pathway.

[0064] 17. Apoptosis test results

[0065] See the instruction manual appendix Figure 17 The results are from the apoptosis assay. PBS was used as the blank control group, and Fe... 3+Toy, Gos, TF, GF, and GT were used as experimental groups. After co-incubating with 4T1 cells in an incubator for 6 hours, the cells were washed three times with PBS, then digested with trypsin, centrifuged at 1000 rpm for 5 minutes to collect the cells, resuspended in PBS, and stained with Annexin V-FITC / PI apoptosis detection reagent for 15 minutes under light-protected conditions. Cell apoptosis was then detected by flow cytometry. (See attached image) Figure 17 As shown, compared to the PBS group (15%), single Fe... 3+ The ability to induce apoptosis in 4T1 cells was limited, with a combined necrosis and apoptosis rate of only 19.3%. Treatment with Toy and Gos increased the combined necrosis and apoptosis rates of 4T1 cells to 36.3% and 38.1%, respectively, indicating that Toy and Gos possess good anticancer activity. Similarly, the combined treatment groups (TF, GF, and GT) further increased the combined necrosis and apoptosis rates of 4T1 cells to 51.8%, 55.4%, and 63.3%, respectively, mainly due to Fe... 3+ The combined effects of Toy-mediated ROS generation, Gos-mediated mitochondrial dysfunction, or Toy-exacerbated endoplasmic reticulum stress were observed. The necrosis rate in the GT group (53.6%) was significantly higher than in other groups, likely due to the dual-pathway chemotherapy mediated by both Toy and Gos, which is related to the presence of... Figure 10 The cytotoxicity results were consistent with those in group A. Furthermore, the combined necrosis and apoptosis rates of the GFT group were 96.62%, indicating that GFT NCAs possess potent anticancer activity.

[0066] 18. Results of in vitro immunogenicity test

[0067] See the instruction manual appendix Figure 18 A and 18B show the results of ATP and HMGB1 release from cells after treatment with different materials, respectively. 4T1 cells were divided into 2 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 6-well cell culture plates and incubated for 24 hours in a 5% CO2, 37°C incubator to allow for cell adhesion and growth. PBS was used as a blank control group, with Fe [missing information - likely a specific ingredient or ingredient]. 3+ Toy, Gos, TF, GF, GT, and GFT NCAs were used as experimental groups. After co-incubating with 4T1 cells in an incubator for 24 hours, the culture medium from the corresponding 6-well plates of each group was collected, and the HMGB1 and ATP content in the culture medium was detected according to the instructions using HMGB1 and ATP assay kits. (See attached...) Figure 18 As shown in Figure A, the level of ATP released by 4T1 cells in the TF, GF, or GT combined treatment group was significantly higher than that in the single treatment group (Fe). 3+(Toy or Gos), indicating that combination therapy significantly enhanced the ICD effect through enhanced mitochondrial dysfunction (GF) or a combination of mitochondrial dysfunction and exacerbated endoplasmic reticulum stress (TF and GT). See attached. Figure 18 As shown in Figure B, compared with the Toy and Gos groups, GT significantly induced the release of HMGB1 in 4T1 cells. Furthermore, GFT NCAs showed the most significant effect in inducing the release of ATP and HMGB1 from 4T1 cells, indicating that GFT NCAs can significantly induce ICD in 4T1 cells.

[0068] See the instruction manual appendix Figure 18 C represents the expression of CRT on the cell membrane surface after treatment with different materials. (1×10) 5 Cells were seeded into laser confocal microscopy dishes and then incubated in a 5% CO2, 37°C cell culture incubator for 24 hours to allow for cell adhesion and growth. PBS was then used as a blank control group, and Fe... 3+ Toy, Gos, TF, GF, GT, and GFT NCAs were used as experimental groups and co-incubated with 4T1 cells in an incubator for 24 h. Cells were then washed three times with PBS and fixed with glutaraldehyde (2.5%) for 10 min. Cells were then treated with immunostaining blocking buffer for 60 min, followed by incubation with anti-CRT (primary antibody) for 60 min. Subsequently, cells were washed with PBS and incubated with Cy3-labeled secondary antibody for 60 min. Finally, before microscopic examination, cells were stained with DAPI at 37°C for 5 min, washed three times with PBS, and observed under CLSM. The results showed that the red fluorescence signal of CRT on the cell membrane surface of the combined treatment group was significantly stronger than that of the single treatment group, and the red fluorescence signal of CRT on the cell membrane surface of the GFT group was the strongest. This further demonstrates that GFTNCAs have a strong ability to induce ICD in cancer cells, which is attributed to the dual-channel induced apoptosis of cancer cells by mitochondrial dysfunction and endoplasmic reticulum stress.

[0069] 19. Results of in vitro dendritic cell maturation (DCs) assay

[0070] See the instruction manual appendix Figure 19 This is the result of in vitro dendritic cell maturation. We used a transwell assay to verify the maturation of dendritic cells induced by tumor cell immunogenic death. First, 4T1 cells were cultured at 1 × 10⁶ cells per well. 5 Cells were seeded at a density of 1 mL of culture medium into the upper chamber of a 6-well plate with a 0.4 μm polycarbonate porous membrane, and incubated in a 5% CO2, 37°C incubator for 12 h to allow cell adhesion and growth. The cell culture medium in the upper chamber was then replaced with a medium containing Fe... 3+Fresh medium containing Toy, Gos, TF, GF, GT, and GFT NCAs was used for further incubation for 24 hours. Meanwhile, DCs were introduced at a rate of 2 × 10⁶ cells per well. 5 The densities of DCs were seeded in 1 mL of culture medium into the wells of the lower chamber and co-incubated with 4T1 cells treated in different ways in the upper chamber for 24 h. Subsequently, the DCs were digested with trypsin and collected by centrifugation. They were then stained with CD86 and CD80 antibodies in the dark for 15 min, respectively. After staining, they were washed with PBS by centrifugation. Finally, the DCs were resuspended in 0.2 mL of PBS for flow cytometry analysis. (See attached image) Figure 19 As shown, Fe 3+ The maturation rates of DCs in the TF (7.93%), Toy (15.9%), and Gos (17.8%) groups were significantly higher than those in the PBS group (1.36%). Furthermore, based on the advantages of combination therapy in inducing ICD in cancer cells, the maturation rates of DCs in the combination therapy groups (TF, GF, and GT) increased to 40.5%, 55.3%, and 66.2%, respectively, and were significantly higher than those in the single-treatment groups (Fe...). 3+ (Toy and Gos). Clearly, due to the significant induction of ICD in cancer cells by GFT NCAs, the maturation rate of DCs in the GFT group was the highest (75.3%). In summary, GFT NCAs effectively promote DC maturation and activate anti-tumor immunity by inducing ICD in 4T1 cells through a dual pathway of inducing mitochondrial dysfunction and exacerbating endoplasmic reticulum stress.

[0071] 20. Results of in vivo T1 magnetic resonance imaging test

[0072] See the instruction manual appendix Figure 20 A and 20B show the in vivo T1 magnetic resonance imaging results and corresponding signal-to-noise ratios (SNR) of tumor-bearing mice. Magnevist mice served as the control group, and GFT NCAs mice served as the experimental group. After equal amounts of material (Gd or Fe) were injected via the tail vein, T1 MRI images of the two groups of mice were recorded at different time points. (See attached image.) Figure 20 As shown, at 15 and 30 minutes after tail vein injection, the T1 MR signal at the tumor site in the Magnevist group was stronger than that in the GFT group, reaching a peak at 30 minutes. The T1 MR signal intensity gradually decreased over time, indicating that Magnevist could reach the tumor site more quickly and be metabolized more rapidly. The T1 MR signal intensity in the GFT group gradually increased over time, reaching a peak at 90 minutes, significantly higher than that in the Magnevist group, and decreased at 105 minutes. In conclusion, GFT NCAs can accumulate at the tumor site, exhibiting sustained T1 MR imaging characteristics at the tumor site, with a significantly higher peak T1 MR signal than Magnevist, and can be metabolized in vivo over time.

[0073] 21. Evaluation of in vivo therapeutic effects

[0074] See the instruction manual appendix Figure 21 A, 21B, and 21C represent the changes in tumor volume, survival rate, and body weight in mice after different treatments, respectively. PBS served as the blank control group, and Gos+Fe... 3+ +Toy (G+F+T), GFT NCAs, and GFT NCAs+A-PD-L1 were used as experimental groups. A 4T1 subcutaneous tumor model was constructed in Balb / c mice, and PBS, G+F+T, and GFT NCAs (100 μL, 30 μg Fe / mouse, 30.6 μg Gos / mouse, or 7.44 g Toy / mouse) were injected intravenously into the tail vein. Mice in the GFT+A-PD-L1 group received an intratumoral injection of A-PD-L1 solution (0.2 mg / mL) one day after the GFT NCAs injection. -1 (100 μL PBS). A total of 4 treatments were administered, once every 3 days. Tumor volume and body weight of each mouse were recorded every two days for 14 days. Survival status of the mice was recorded every two days for 30 days. (See attached...) Figure 21 As shown in Figure A, compared with the PBS group, the free drug group G+F+T showed a certain tumor-suppressive effect, mainly due to the chemotherapeutic effect of the drug and Fe. 3+ While CDT mediated tumor suppression, drug accumulation at the tumor site was limited. Compared to the G+F+T group, GFT NCAs showed more significant tumor suppression, mainly due to EPR-mediated enrichment of GFT NCAs at the tumor site. Further combination with ICB therapy resulted in the most significant tumor suppression effect from the GFT NCAs + A-PD-L1 mediated trimodal therapy, and also exhibited the highest survival rate (100%) in mice 30 days after treatment (see appendix). Figure 21 (B) This further demonstrates the potent antitumor efficacy of the combination therapy of GFT NCAs and A-PD-L1. Conversely, the survival rates of mice in the PBS and G+F+T groups were less than or equal to 60%, significantly lower than those in the GFT+A-PD-L1 group. Furthermore, the stable changes in mouse body weight after different treatments throughout the experiment indicate that the combination therapy of GFT NCAs and A-PD-L1 has good biocompatibility (see appendix). Figure 21 C).

[0075] Beneficial effects

[0076] (1) The reaction conditions of this invention are easy to achieve and highly controllable. By precisely controlling the reaction parameters and synthesis conditions, the spatiotemporal separation of nanocapsules during the formation process along the microfluidic channel is achieved, thereby obtaining nanocapsules with ideal size, morphology, and composition. Microfluidic synthesis also has the advantages of low reagent consumption and continuous production, and has good development prospects in the field of nanomedicine preparation.

[0077] (2) The gossypol-ferric ion-fumigin iron polyphenol nanocapsules (GFT NCAs) prepared in this invention have simple components, good stability, and pH-responsive properties. In the microacidic environment of tumors, GFT NCAs can release Gos, Fe3+, and Toy. Based on the synergistic effect of Gos-induced mitochondrial dysfunction, Fe3+-mediated CDT, and Toy-exacerbated endoplasmic reticulum stress, GFT NCAs can significantly induce apoptosis and immunogenic death in cancer cells, providing a new design concept for constructing safe and efficient simple-component nanomedicines.

[0078] (3) The gossypol-trivalent iron ion-Fungamicin iron polyphenol nanocapsules (GFT NCAs) prepared in this invention can not only achieve T1 MRI of the tumor site after being injected into mice via the tail vein, but also combine with immune checkpoint blockade to achieve a three-modal combined treatment of chemokine therapy / dual-channel chemotherapy / immunotherapy, further enhancing the anti-tumor effect and having potential clinical application value.

[0079] (4) The present invention solves the problems of difficult control of the preparation process, uneven product size and poor repeatability of traditional wet chemical methods. Attached Figure Description

[0080] Figure 1 This diagram illustrates the synthesis and application of the GFT NCAs prepared in this invention.

[0081] Figure 2 Hydrodynamic diameter and surface potential diagram of GFT NCAs prepared in this invention in water;

[0082] Figure 3 A graph showing the hydrodynamic diameter of the GFT NCAs prepared in this invention as a function of time in PBS solution (pH = 7.4);

[0083] Figure 4 Ultraviolet (A) and infrared (B) spectra of Gos, Toy, and GFT NCAs;

[0084] Figure 5 XPS spectrum (A) and high-resolution Fe 2p XPS spectrum (B) of the GFT NCAs prepared for this invention;

[0085] Figure 6 TEM images (A and B) of the GFT NCAs prepared in this invention;

[0086] Figure 7 T1 MRI images (A) and relaxation rate r1 (B) of GFT NCAs prepared for this invention under different pH conditions;

[0087] Figure 8 MB degradation performance analysis of GFT NCAs prepared for this invention: MB degradation curves under different conditions (A), UV spectrum and solution photograph of MB degradation of GFT NCAs in a slightly acidic phosphate buffer solution (pH=6.5) containing H2O2 (B);

[0088] Figure 9 Release curves of Gos(A), Fe(B) and Toy(C) of the GFT NCAs prepared for this invention in phosphate buffers at different pH values;

[0089] Figure 10 For Gos, Toy, Fe 3+ Cytotoxicity analysis of TF, GF, GT and GFT NCAs: (A) Cell viability graphs of 4T1 cells after 24 h of treatment with different concentrations of Gos or Toy, Gos, Toy, GT or GFT NCAs; (B) Cell viability graphs of different Fe 3+ Concentration of Fe 3+ Cell viability of 4T1 cells after 24 hours of treatment with TF, GF or GFT NCAs;

[0090] Figure 11 For 4T1 cells to react with GFT NCAs and Fe 3+ Intake status;

[0091] Figure 12 Evaluation of the hemolysis rate of erythrocytes in Balb / c mice using GFT NCAs prepared in this invention;

[0092] Figure 13 For PBS, Toy, Gos, Fe 3+ Effects of GT, TF, GF and GFT NCAs on intracellular ROS in 4T1 cells (A) and GSH (B);

[0093] Figure 14 For PBS, Toy, Gos, Fe 3+ Effects of GT, TF, GF and GFT NCAs on LPO content in 4T1 cells, scale bar 20 μm;

[0094] Figure 15 For PBS, Fe 3+Effects of Toy, Gos, TF, GT, GF and GFT NCAs on mitochondrial membrane potential in 4T1 cells, with a scale bar of 20 μm;

[0095] Figure 16 For PBS, Fe 3+ Western blot analysis results of endoplasmic reticulum stress-related proteins GRP78, p-IRE1α, XBP1u, XBP1s and CHOP in 4T1 cells using Gos, Toy, GF, TF, GT and GFT NCAs;

[0096] Figure 17 For PBS, Fe 3+ Effects of Toy, Gos, TF, GF, GT and GFT NCAs on apoptosis in 4T1 cells;

[0097] Figure 18 For PBS, Fe 3+ Evaluation of immunogenic cell death induced by Toy, Gos, TF, GF, GT and GFT NCAs: ATP release (A), HMGB1 release (B), and expression of CRT on the surface of 4T1 cell membrane (C).

[0098] Figure 19 For PBS, Fe 3+ Evaluation of how Toy, Gos, TF, GF, GT, and GFT NCAs induce dendritic cell maturation through immunogenic death of 4T1 cells;

[0099] Figure 20 T1 MRI images (A) and corresponding SNR (B) of tumors in tumor-bearing mice at different time points before and after tail vein injection of GFT NCAs and the clinical T1 MRI contrast agent Magnevist prepared for this invention.

[0100] Figure 21 The figures show the relative tumor volume changes (A), survival rate changes (B), and body weight changes (C) of each group of mice. Detailed Implementation

[0101] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims. Polydimethylsiloxane (PDMS) was purchased from Dow Corning, Inc., USA. Ferrous chloride tetrahydrate was purchased from Adamas Reagents Ltd. (Shanghai, China). Gossypol was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Fungamicin was purchased from Shanghai Weihuan Biotechnology Co., Ltd. (Shanghai, China). DMEM culture medium, fetal bovine serum (FBS, GIBCO), penicillin-streptomycin (HyClone, Thermo Scientific, Logan, UT), and trypsin 0.25% solution (HyClone) were purchased from Hangzhou Gino Biomedical Technology Co., Ltd. (Hangzhou, China). C11 BODIPY 581 / 591 All cells were purchased from Shanghai Maokang Biotechnology Co., Ltd. (Shanghai, China). Cell Counting Kit-8 (CCK-8) and Annexin V-FITC / PI apoptosis detection kit were purchased from 7Sea Biotech Co., Ltd. (Shanghai, China). GSH detection kit was purchased from Nanjing Jiancheng Biotechnology Co., Ltd. (Nanjing, China). ROS detection kit was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). 4T1 cells (mouse breast cancer cell line) were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. 4-5 week old Balb / c mice were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd. (Shanghai, China). All water with a resistivity higher than 18.2 MΩ·cm used in the experiments was purified through a laboratory water purification system (Cascada I, PALL, Beijing, China).

[0102] Example 1

[0103] (1) The microfluidic channel structure was designed using AutoCAD software. The microfluidic channel design adopted an S-shaped channel, including: five inlets, one outlet, and an S-shaped channel. The height of each channel was 50 μm. The width of the microfluidic channels at inlets 1, 2, and 3 was 100 μm, and the width of the remaining microfluidic channels was 300 μm. The total length from the channel inlet to the outlet was 34.6 mm (e.g., ...). Figure 1(As shown); then, the designed chip is printed into a mask using a high-resolution printer, and a microfluidic chip mold is fabricated on a silicon wafer using photolithography. Finally, the fabricated chip mold is used to cast the corresponding polydimethylsiloxane (PDMS) microfluidic channel cover sheet; using plasma bonding technology, with a vacuum of 20 Pa and treatment in an air atmosphere for 80 s, a glass slide is used as a substrate and bonded to the aforementioned PDMS microfluidic channel cover sheet to obtain the microfluidic chip.

[0104] (2) Mix ferric chloride ethanol solution (8.11 mg / mL, 12.8 μL) and gossypol ethanol solution (7.78 mg / mL, 128 μL), then add ultrapure water (19.2 μL), and sonicate for 3 min to obtain a mixed solution of gossypol iron intermediate (6.87 mg / mL, 0.16 mL); mix ferric chloride ethanol solution (8.11 mg / mL, 38.4 μL) and fugamycin ethanol solution (0.87 mg / mL, 640 μL), then add ultrapure water (4121.6 μL), and sonicate for 3 min to obtain a mixed solution of fugamycin iron intermediate (0.18 mg / mL, 4.8 mL).

[0105] (3) The mixed solution of ferric intermediate in step (2) was injected into the injection port 1 (first injection port) by a micro-injection pump at a flow rate of 4.8 mL / h; the mixed solution of gossypol iron intermediate in step (2) was injected into the injection port 2 (second injection port) by a micro-injection pump at a flow rate of 0.16 mL / h; Tris buffer (pH=10.5) was injected into the injection port 3 (third injection port) by a micro-injection pump at a flow rate of 49.6 mL / h; Tris buffer (pH=10.5) was injected into the injection port 4 (fourth injection port) by a micro-injection pump at a flow rate of 4.8 mL / h, and collected through the outlet port 5 to obtain gossypol-ferric ion-ferric polyphenol nanocapsules (GFT NCAs).

[0106] Example 2

[0107] The GFT NCAs prepared in Example 1 were characterized. The hydrodynamic diameter and surface potential of the GFT NCAs are as follows: Figure 2 As shown, the potential of GFT NCAs is -22.3±0.5mV, the hydrated particle size is 185.5±2.9nm, and the PDI is 0.271±0.042, indicating that GFT NCAs have good dispersibility and suitable size. Figure 3 As shown, the hydrated particle size of GFT NCAs did not change significantly during the one-week monitoring period and remained close to its initial hydrated particle size (185.5 ± 2.9 nm), indicating that GFT NCAs have good colloidal stability.

[0108] like Figure 4 As shown in Figure A, the absorption peaks at 234 nm and 374 nm are characteristic peaks of Gos, while the absorption peak at 279 nm is a characteristic peak of Toy. A new absorption peak appears at 251 nm in GFT NCAs, and the characteristic peak of Gos at 374 nm in GFT NCAs redshifts to 383 nm. These findings are attributed to the interaction of Gos and Fe. 3+ The coordination between Gos-Fe and Toy indicates the formation of Gos-Fe 3+ -Toy's metal polyphenol network structure. Furthermore, GFT NCAs exhibit a new absorption band in the 500-800 nm wavelength range, attributed to Gos-Fe. 3+ The aggregated structure of -Toy demonstrates the successful preparation of GFT NCAs.

[0109] like Figure 4 As shown in B, 597cm -1 The absorption peak at 2961 cm⁻¹ is a characteristic peak of the Fe-O bond in GFT NCAs; -1 and 1380cm -1 These are the characteristic peaks of the CH bond stretching vibration and bending vibration of the methyl group in Gos, respectively, at 1455 cm⁻¹. -1 This is the characteristic peak of the C=C stretching vibration of the benzene ring in Gos; 1025 cm⁻¹ -1 The peak represents the stretching vibration characteristic of the CN bond in Toy. This indicates the presence of the Gos, Toy, and Fe-O coordination structure in GFT NCAs, further proving the successful preparation of GFT NCAs.

[0110] Figure 5 A and 5B are the XPS spectra of GFT NCAs and the high-resolution Fe 2p XPS spectra, respectively. Figure 5 As shown in Figure A, four elements appeared in the XPS spectra of GFT NCAs, including Fe, O, C, and N. The characteristic peak of Fe originated from Fe. 3 + The characteristic peaks of N originate from Toy, while the characteristic peaks of O and C are attributed to Gos and Toy. High-resolution XPS spectra of Fe 2p in GFT NCAs ( Figure 5 B) can be fitted with four photoelectron peaks at 711.69, 725.51, 717.54, and 732.42 eV, corresponding to Fe2p, respectively. 3 / 2 Fe 2p 1 / 2 Fe 2p 3 / 2 satellite and Fe 2p 1 / 2 The binding energy of the satellite proves the Fe in GFT NCAs. 3+ The existence of.

[0111] Figure 6 A and 6B are TEM images of GFT NCAs. GFT NCAs are regular nanocapsules with uniform size and an average particle size of 101.9 ± 7.4 nm.

[0112] Example 3

[0113] The Fe content in GFT NCAs was determined by ICP-OES. To test the T1 MRI performance of GFT NCAs, a series of concentration gradient solutions (1 mL each) were prepared using PBS buffer (Fe concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 mM). The T1 relaxation time of each GFT NCA was measured, and the reciprocal of the relaxation time was linearly fitted to the Fe concentration; the slope of this linear fit was the relaxation rate (e.g., ...). Figure 7 (The relaxation rate of T1 imaging is represented by r1). From the figure, it can be seen that the r1 of GFT NCAs is 2.76 mM. -1 s -1 To test the acid-responsive T1 MRI performance of GFT NCAs, a series of concentration gradient solutions (1 mL each) were prepared using slightly acidic phosphate buffer (pH 6.5) with Fe element concentrations of 0.1, 0.2, 0.4, 0.8, and 1.6 mM. The T1 relaxation times of the GFT NCAs in the weakly acidic buffer solutions were measured, and the reciprocal of the relaxation time was linearly fitted against the Fe concentration; the slope of this fit was the relaxation rate. Figure 7 As shown, in a weakly acidic buffer solution, the r1 of GFTNCAs increased to 3.71 mM. -1 s -1 This indicates that GFT NCAs have good acid-responsive enhanced T1MRI performance and can be used as a good T1 MRI contrast agent in MRI molecular imaging diagnosis.

[0114] GFT NCAs' ROS generation capability, such as Figure 8 As shown, Figure 8 A represents the degradation of methylene blue (MB) under different conditions. Under different conditions, MB degraded to varying degrees within 110 min. Under physiological conditions in the absence of GFT NCAs (pH = 7.4), only a small amount of MB degraded regardless of the presence of H₂O₂. Under physiological conditions containing H₂O₂, the degradation rate of MB reached 20.7% after 110 min, which is attributed to the small amount of Fe released by GFT NCAs under physiological conditions. 3+Furthermore, under slightly acidic conditions containing both GFT NCAs and H2O2, the degradation rate of MB increased to 52.6% after 110 min, which is 2.5 times the MB degradation rate of GFTNCAs under physiological conditions. This is because the Fe in GFT NCAs increases under slightly acidic conditions. 3+ A large amount of MB was released. From the actual images of MB degradation, a significant lightening of MB color can also be observed, indicating that a large amount of MB was degraded. In summary, based on Fe... 3+ GFT NCAs exhibit excellent ROS generation performance in slightly acidic TME containing H2O2 via H2O2-mediated Fenton-like reactions.

[0115] Two PBS buffer solutions with pH=7.4 and pH=6.5 were prepared separately. The prepared GFT NCAs were dissolved in 1 mL of each buffer solution to prepare a solution with a Fe concentration of 200 μg / mL. This solution was then placed in a dialysis bag, which was then placed in a container containing 9 mL of each of the two buffer solutions and shaken in a constant temperature shaker at 37°C. At different time points, 1 mL of the dialysis bag fluid was aspirated, and an equal volume of corresponding PBS buffer solution was added to the container. 0.5 mL of the dialysis bag fluid was digested with aqua regia, and the Fe content was determined by ICP-OES. The remaining 1 mL was filtered and analyzed by a UV-Vis spectrophotometer. The UV absorbance at 279 nm and 383 nm wavelengths in the samples taken at each time point was recorded. After the sustained-release phase, drug release curves of the GFT NCAs under different conditions were plotted. Figure 9 As shown, under weakly acidic conditions (pH 6.5), after 96 hours, the cumulative release of Gos, Fe, and Toy in GFT NCAs reached 16.9%, 14.2%, and 62.9%, respectively. These figures were 3.0, 2.2, and 3.3 times higher than the cumulative release of Gos (5.7%), Fe (6.4%), and Toy (19.0%) under physiological conditions (pH 7.4), respectively. This indicates that GFT NCAs can responsively release Gos, Fe, and Toy under acidic TME conditions to achieve highly efficient and specific tumor therapy.

[0116] Example 4

[0117] Using 4T1 cells as a cell model to evaluate Gos, Toy, and Fe 3+ Gos+Toy (GT), Gos+Fe 3+ (GF), Toy+Fe 3+ (TF) and GFT NCAs cytotoxicity. 4T1 cells were cultured at 1×10 4Cells were seeded at a density of 100 cells per well in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours. The culture medium in the 96-well plates was then replaced with media containing Gos, Toy, and Fe. 3+ Cells were cultured with GT, GF, TF, and GFT NCAs in 5% CO2 at 37°C for 24 hours. For 96-well cell culture plates, the cells were washed three times with PBS, then 100 μL / well of DMEM medium containing 10% (v / v) CCK-8 (10 μL) was added, and the plates were incubated for another 4 hours. The absorbance of each well was measured at 450 nm using a microplate reader. Cells treated with DMEM medium served as a blank control, and cell viability was labeled as 100%. Figure 10 As shown in Figure A, 4T1 cells treated with different methods all exhibited [Gos] or [Toy]-dependent cell viability decreases, and the half-inhibitory concentrations (IC50) of GT and GFT NCAs on 4T1 cells were calculated. 50 The concentrations (s) were 2.8 and 4.4 μg·mL, respectively. -1 ([Toy]), or 10.1 and 18.1 μg·mL, respectively. -1 ([Gos]), where the individual Toy and Gos ICs 50 s were 3.4 μg·mL -1 ([Toy]) and 15.6 μg·mL -1 ([Gos]). Clearly, GT([Toy] = 2.48 μg·mL -1 Or [Gos] = 10.2 μg·mL -1 GT showed stronger cytotoxicity than Gos or Toy alone, demonstrating the superior anticancer activity of dual-pathway chemotherapy. At this concentration, GT exhibited higher cytotoxicity than GFT NCAs, which may be related to the release of Gos, Toy, and Fe from GFT NCAs in 4T1 cells. 3+ It is related to the quantity. For example, Figure 10 As shown in Figure B, 4T1 cells treated differently also exhibited [Fe 3+ [Dependent decrease in cell viability, however, Fe alone] 3+ The anticancer activity was limited, and further calculations showed that the IC50 of GF, TF, and GFT NCAs against 4T1 cells was... 50 The values ​​of s were 13.7, 12.5, and 17.5 μg·mL, respectively. -1 ([Fe 3+ It can be observed that by jointly introducing Toy and Fe... 3+ In combination chemotherapy / CDT therapy, TF showed higher cytotoxicity than GF, which may be due to the stronger cytotoxicity of TF and the effect of chemotherapy combined with CDT. When [Fe 3+= 20.0 μg·mL -1 At this concentration, TF showed stronger cytotoxicity than GFT NCAs, primarily attributed to the drug release efficiency of GFT NCAs in 4T1 cells. At this concentration, based on combination therapy with chemotherapy and CDT, GFT NCAs exhibited better cytotoxicity than Fe. 3+ Much higher cytotoxicity. Overall, GFTNCAs exhibit good antitumor activity.

[0118] like Figure 11 The image shows the reaction of 4T1 cells to GFT NCAF and free Fe. 3+ Phagocytosis. 4T1 cells were divided into groups of 1×10... 5 Cells were seeded at a density of 100 cells per well in 12-well plates and incubated at 5% CO2 and 37°C for 24 h. Then, at 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h, the medium in the corresponding wells of the 12-well plates was replaced with medium containing GFT NCA (Fe concentration set at 20 μg / mL). The cells were then co-cultured with the medium at 5% CO2 and 37°C for another 12 h. After washing three times with PBS, the cells were digested with 500 μL of aqua regia. The solution was diluted with ultrapure water, and the Fe content was determined by ICP-OES. Observations showed that GFT NCAs or Fe... 3+ The incubated cells all exhibited time-dependent cellular uptake. Furthermore, with free Fe... 3+ Compared with the control group, the iron uptake in the GFT group was significantly higher after co-incubation with cells for 4 hours, indicating that GFTNCAs with nanoscale size are more conducive to the phagocytosis of cancer cells.

[0119] like Figure 12 The hemolysis of GFT NCAs is shown. Water was used as the positive control group, and different concentrations of GFT NCAs were used as experimental groups. Each material was co-incubated with Balb / c mouse erythrocyte suspension for 2 hours. UV-Vis absorbance analysis revealed that the hemolysis rate of all GFT NCAs groups was less than the threshold of 5%, while the hemolysis rate of the positive control group was significantly higher, indicating that GFT NCAs have good blood compatibility.

[0120] Example 5

[0121] Evaluation of Toy, Gos, and Fe using 4T1 cells 3+ The effects of GT, TF, GF, and GFT NCAs on intracellular ROS levels. 4T1 cells were seeded in four 6-well plates (2 × 10⁶ cells / well). 5 (Number of cells / well) incubated overnight (37°C, 5% CO2). Remove the old culture medium and replace it with medium containing Toy, Gos, and Fe. 3+Cells were incubated with DMEM medium containing GT, TF, GF, or GFT NCAs ([Fe] = 20 μg / mL, [Gos] = 20.4 μg / mL, and [Toy] = 4.96 μg / mL) for 6 h (37℃, 5% CO2), with the PBS group serving as a control. After washing three times with PBS, 2000 μL of DMEM medium and 2 μL of 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) were slowly added to each well under light-protected conditions, and incubation was continued for 30 min (37℃, 5% CO2). After PBS washing, cells were digested with trypsin, centrifuged at 1000 rpm for 5 min to collect the cells, resuspended in PBS, and intracellular ROS levels were detected by flow cytometry. Figure 13 As shown in Figure A, compared to the PBS control group, the use of Toy, Gos, or Fe... 3+ ROS levels in the treated cells were significantly improved. Toy amplified the endoplasmic reticulum stress effect in cancer cells, thereby affecting intracellular ROS levels. Similarly, Gos increased intracellular ROS levels, decreased mitochondrial membrane potential, caused mitochondrial dysfunction, and induced oxidative stress in cancer cells. 3+ On one hand, it undergoes a Fenton-like reaction with intracellular H2O2, thereby producing ROS and Fe. 2+ Fe 2+ The Fenton-mediated response further increased intracellular ROS levels. Furthermore, the intracellular ROS levels in the combination therapy groups (GT, TF, and GF) were significantly higher than those in the single therapy groups (Toy, Gos, and Fe). 3+ This indicates that combined therapy is more conducive to the generation of intracellular ROS. Of course, GFT NCAs exhibited the strongest ROS generation capacity across all groups, primarily due to Toy, Gos, and Fe... 3+ The three have a synergistic promoting effect on intracellular ROS generation.

[0122] Evaluation of Toy, Gos, and Fe using 4T1 cells 3+ The effects of GT, TF, GF, and GFT NCAs on intracellular GSH levels. First, 4T1 cells were seeded into four 6-well plates (2 × 10⁶ cells / wells). 5 (samples / well), incubated overnight in a constant temperature incubator (37℃, 5% CO2). Remove the old culture medium and replace it with media containing Toy, Gos, and Fe. 3+Cells were incubated in a medium containing GT, TF, GF, or GFT NCAs ([Fe] = 20 μg / mL, [Gos] = 20.4 μg / mL, and [Toy] = 4.96 μg / mL) for 6 hours (37℃, 5% CO2), with the PBS group serving as a control. Cells were slowly washed with PBS, digested, collected, resuspended in PBS, and centrifuged again to collect more cells. Intracellular GSH levels were detected using a GSH assay kit. The experimental results are shown below. Figure 13 As shown in Figure B, compared to the PBS group, the other groups all showed varying degrees of GSH depletion, corresponding to the ROS generation capacity mentioned earlier. Among them, Fe... 3+ It exhibits good GSH consumption capacity, which is due to Fe 3+ It can be reduced to Fe by high concentrations of GSH. 2+ And Fe 2+ The ROS generated by the Fenton-mediated reaction can further consume GSH and regenerate Fe. 3+ Interestingly, Fe 3+ and Fe 2+ The classic cyclical response between these factors can lead to sustained GSH depletion and ROS generation, thereby modulating the TME. Similarly, ROS generation mediated by combination therapies (GT, TF, and GF) also exhibits significantly higher GSH depletion capacity than the single-treatment groups (Toy, Gos, and Fe). 3+ Furthermore, the GFT group exhibited the highest GSH consumption, with an intracellular GSH content of only 20.23%. This was attributed to the promoting effect of the combination therapy on intracellular ROS generation and the presence of Fe... 3+ and Fe 2+ The classic cyclic reaction between them mediates continuous TME regulation. In summary, GFT NCAs possess excellent TME regulation capabilities.

[0123] Example 6

[0124] Evaluation of Toy, Gos, and Fe using 4T1 cells 3+ The effects of GT, TF, GF, and GFT NCAs on intracellular LPO levels. 4T1 cells were seeded in eight confocal dishes (1×10⁻⁶). 5 (samples / well), incubated overnight in a constant temperature incubator (37℃, 5% CO2). Remove the old culture medium and replace it with media containing Toy, Gos, and Fe. 3+Fresh medium containing GT, TF, GF, or GFT NCAs ([Fe] = 20 μg / mL, [Gos] = 20.4 μg / mL, and [Toy] = 4.96 μg / mL) was used for incubation for 6 h (37℃, 5% CO2), with the PBS group serving as a control. After slow washing with PBS, 500 μL of LMEM medium and 1 μL of C11 BODIPY were slowly added to each well under dark conditions. After co-culturing for 20 min, the cells were washed three times slowly with PBS, fixed with glutaraldehyde (2.5%) for 10 min, stained with DAPI for 5 min, and finally recorded the blue, red, and green fluorescence signals of the cells after different treatments using CLSM. Figure 14 As shown, compared with the PBS control group, all other groups exhibited enhanced green fluorescence signal (oxidized C11-BODIPY). 581 / 591 ) and the correspondingly weakened red fluorescence signal (non-oxidized C11-BODIPY) 581 / 591 ), indicating Toy, Gos, Fe 3+ GT, TF, GF, and GFT NCAs can all induce LPO accumulation in cancer cells. Compared with the PBS, Toy, and Gos groups, Fe... 3+ The red fluorescence signal of the treated cells was significantly reduced, while the green fluorescence signal was significantly enhanced. This is attributed to Fe. 3+ Mediated ROS generation and GSH consumption. Furthermore, compared to single treatment groups (Toy, Gos, and Fe) 3+ Compared to the GT, TF, and GF groups, the cells showed a further decrease in red fluorescence intensity and a corresponding increase in green fluorescence intensity, indicating that the combined treatment significantly promoted intracellular LPO accumulation. Furthermore, based on the combined treatment-mediated ROS generation and GSH consumption, the LPO accumulation effect was most significant in the GFT group.

[0125] Example 7

[0126] Evaluation of Fe using 4T1 cells 3+ The effects of Toy, Gos, TF, GT, GF, and GFT NCAs on mitochondrial membrane potential. 4T1 cells were seeded in eight confocal dishes (1×10⁻⁶). 5 (samples / well), incubated overnight in a constant temperature incubator (37℃, 5% CO2). Remove the old culture medium and replace it with media containing Toy, Gos, and Fe. 3+Fresh medium containing GT, TF, GF, or GFT NCAs ([Fe] = 20 μg / mL, [Gos] = 20.4 μg / mL, and [Toy] = 4.96 μg / mL) was added and incubated for 6 h (37℃, 5% CO2), with the PBS group serving as a control. After slow rinsing with PBS, 1 mL of JC-1 staining working solution and 1 mL of fresh medium were slowly added to each well under dark conditions. After mixing, the cells were co-cultured for 20 min. The supernatant of old medium was removed, and the cells were gently rinsed twice with JC-1 staining buffer. 2 mL of LDM medium was added to each well, and the red and green fluorescence signals of the cells were observed using CLSM. Figure 15 As shown, compared with the PBS control group, Fe 3+ The mitochondria of cancer cells in the Toy and Gos groups showed green fluorescence, indicating that Fe... 3+ Toy and Gos can all reduce mitochondrial membrane potential to varying degrees, possibly due to Fe... 3+ The effects of Toy-mediated ROS generation, Toy-amplified endoplasmic reticulum stress on mitochondrial membrane potential, and Gos-mediated mitochondrial dysfunction. Compared to the single-treatment group (Fe... 3+ The combination therapy group (including Toy and Gos) showed stronger green fluorescence and correspondingly weaker red fluorescence. The GFT group exhibited the weakest red fluorescence and the strongest green fluorescence, indicating that GFT NCAs can induce significant mitochondrial dysfunction and help induce apoptosis in cancer cells.

[0127] Example 8

[0128] Evaluation of Fe using 4T1 cells 3+ Effects of Toy, Gos, TF, GT, GF, and GFT NCAs on endoplasmic reticulum stress. 4T1 cells were seeded into 6-well plates (2 × 10⁶ cells / well). 5 (each well contains 10 cells / well), and incubate overnight in a constant temperature incubator (37°C, 5% CO2). Then, replace the old culture medium with one containing Fe. 3+ Fresh medium containing Toy, Gos, TF, GT, GF, or GFT NCAs ([Fe] = 20 μg / mL, [Gos] = 20.4 μg / mL, and [Toy] = 4.96 μg / mL) was used for incubation for 24 h (37℃, 5% CO2), with the PBS group serving as a control. The medium was discarded, and the cells were slowly washed three times with PBS, followed by trypsin digestion to collect the cells. Cells were then lysed and proteins extracted. Protein concentration was determined, and proteins were separated by SDS-PAGE electrophoresis. Cells were then sequentially transferred, blocked, and incubated with primary and secondary antibodies. Imaging was performed using a chemiluminescence / fluorescent gel imaging system, and finally, protein content was quantified using ImageJ. Figure 16As shown, after treatment with different materials, the expression levels of the endoplasmic reticulum stress marker protein GRP78 and the p-IRE1α protein related to the endoplasmic reticulum stress homeostasis recovery pathway p-IRE1α-XBP1 were upregulated to varying degrees. This is due to Fe 3+ Gos can disrupt the redox balance in cancer cells, inducing oxidative stress and thus exacerbating endoplasmic reticulum (ER) stress. Toy, on the other hand, inhibits the restoration of ER homeostasis by blocking the p-IRE1α-XBP1 pathway, thereby inducing increased ER stress. Interestingly, both Toy and Toy-related groups showed upregulation of XBP1u protein expression and downregulation of the corresponding XBP1s protein expression. This is attributed to Toy blocking the p-IRE1α-XBP1 pathway, preventing p-IRE1α from cleaving XBP1u, thus reducing XBP1s expression and leading to further upregulation of XBP1u expression. Moreover, GFT NCAs induced the most significant upregulation of GRP78, p-IRE1α, and XBP1u, and the most significant downregulation of XBP1s, indicating that GFT NCAs have the ability to significantly exacerbate ER stress in cancer cells. Based on this, the expression levels of CHOP, a marker protein of apoptosis induced by ER stress pathways in cancer cells under different treatments, were further investigated. Cancer cells treated with different methods all showed varying degrees of CHOP upregulation, with GFT NCAs inducing the most significant CHOP upregulation, indicating that GFT NCAs can significantly induce cancer cell apoptosis through the endoplasmic reticulum stress pathway.

[0129] Example 9

[0130] Evaluation of Fe using 4T1 cells 3+ Effects of Toy, Gos, TF, GF, GT, and GFT NCAs on apoptosis. 4T1 cells were seeded in four 6-well plates (2 × 10⁶ cells / well). 5 (samples / well), incubated overnight in a constant temperature incubator (37℃, 5% CO2). Remove the old culture medium and replace it with media containing Toy, Gos, and Fe. 3+ Fresh medium containing GT, TF, GF, or GFT NCAs ([Fe] = 20 μg / mL, [Gos] = 20.4 μg / mL, and [Toy] = 4.96 μg / mL) was incubated for 6 h (37℃, 5% CO2), with the PBS group serving as a control. Cells were slowly washed three times with PBS, digested with trypsin, and collected (1000 rpm, 5 min). The cells were resuspended in PBS, and under light-protected conditions, 5 μL of Annexin V-FITC was added to each tube. The reaction was carried out at room temperature for 10 min, followed by the addition of 10 μL of LPI staining solution, and the reaction was carried out at room temperature for 5 min. Apoptosis levels were then detected by flow cytometry. Figure 17 As shown, compared to the PBS group (15%), single Fe... 3+The ability to induce apoptosis in 4T1 cells was limited, with a combined necrosis and apoptosis rate of only 19.3%. Treatment with Toy and Gos increased the combined necrosis and apoptosis rates of 4T1 cells to 36.3% and 38.1%, respectively, indicating that Toy and Gos possess good anticancer activity. Similarly, the combined treatment groups (TF, GF, and GT) further increased the combined necrosis and apoptosis rates of 4T1 cells to 51.8%, 55.4%, and 63.3%, respectively, mainly due to Fe... 3+ The combined effects of Toy-mediated ROS generation, Gos-mediated mitochondrial dysfunction, or Toy-exacerbated endoplasmic reticulum stress were observed. The necrosis rate in the GT group (53.6%) was significantly higher than in other groups, likely due to the dual-pathway chemotherapy mediated by both Toy and Gos. Figure 10 The cytotoxicity results were consistent with those in group A. Furthermore, the combined necrosis and apoptosis rates of the GFT group were 96.62%, indicating that GFT NCAs possess potent anticancer activity.

[0131] Example 10

[0132] Using 4T1 cells as a cell model to evaluate Fe 3+ Effects of Toy, Gos, TF, GF, GT, and GFT NCAs on ATP and HMGB1 release from cells. 4T1 cells were seeded in 6-well plates (2 × 10⁻⁶). 5 (samples / well), incubated overnight in a constant temperature incubator (37℃, 5% CO2). Remove the old culture medium and replace it with media containing Toy, Gos, and Fe. 3+ Fresh medium containing GT, TF, GF, or GFT NCAs ([Fe] = 20 μg / mL, [Gos] = 20.4 μg / mL, and [Toy] = 4.96 μg / mL) was used for co-culturing for 24 h (37℃, 5% CO2), with the PBS group serving as a control. Subsequently, the culture medium from each well was collected, and the levels of HMGB1 and ATP in the culture medium were measured according to the kit instructions using an HMGB1 and ATP assay kit. Figure 18 As shown in Figure A, the level of ATP released by 4T1 cells in the TF, GF, or GT combined treatment group was significantly higher than that in the single treatment group (Fe). 3+ (Toy or Gos), indicating that combination therapy significantly enhanced the ICD effect through enhanced mitochondrial dysfunction (GF) or a combination of mitochondrial dysfunction and exacerbated endoplasmic reticulum stress (TF and GT). Figure 18 As shown in Figure B, compared with the Toy and Gos groups, GT significantly induced the release of HMGB1 in 4T1 cells. Furthermore, GFT NCAs showed the most significant effect in inducing the release of ATP and HMGB1 from 4T1 cells, indicating that GFT NCAs can significantly induce ICD in 4T1 cells.

[0133] Using 4T1 cells as a cell model to evaluate Fe 3+ The effects of Toy, Gos, TF, GF, GT, and GFT NCAs on CRT expression on the cell membrane surface. 1×10 5 Cells were seeded into laser confocal microscopy dishes and then incubated in a 5% CO2, 37°C cell culture incubator for 24 hours to allow for cell adhesion and growth. PBS was then used as a blank control group, and Fe... 3+ Toy, Gos, TF, GF, GT, and GFT NCAs were used as experimental groups and co-incubated with 4T1 cells in an incubator for 24 h. The cells were then washed three times with PBS and fixed with glutaraldehyde (2.5%) for 10 min. Next, the cells were treated with immunostaining blocking buffer for 60 min, followed by incubation with anti-CRT (primary antibody) for 60 min. Subsequently, the cells were washed with PBS and incubated with Cy3-labeled secondary antibody for 60 min. Finally, before microscopic examination, the cells were stained with DAPI at 37°C for 5 min, washed three times with PBS, and observed under CLSM. Figure 18 As shown in Figure C, the red fluorescence signal of CRT on the cell membrane surface of the combined treatment group was significantly stronger than that of the single treatment group, and the red fluorescence signal of CRT on the cell membrane surface of the GFT group was the strongest, further demonstrating that GFT NCAs have a strong ability to induce ICD in cancer cells. This is attributed to the dual-channel induced apoptosis of cancer cells caused by mitochondrial dysfunction and endoplasmic reticulum stress. In summary, this indicates that the combined treatment of chemokinetic therapy / dual-channel chemotherapy has the most significant effect in inducing immunogenic cell death in 4T1 cells.

[0134] Using 4T1 cells as a cell model to evaluate Fe 3+ Toy, Gos, TF, GF, GT, and GFT NCAs affect DC maturation by inducing immunogenic cell death in tumor cells. Transwell assays were used to verify the immunogenic cell death-induced DC maturation. First, 4T1 cells were cultured at 1 × 10⁶ cells per well. 5 Cells were seeded at a density of 1 mL of culture medium into the upper chamber of a 6-well plate with a 0.4 μm polycarbonate porous membrane, and incubated in a 5% CO2, 37°C incubator for 12 h to allow cell adhesion and growth. The cell culture medium in the upper chamber was then replaced with a medium containing Fe... 3+ Fresh medium containing Toy, Gos, TF, GF, GT, and GFT NCAs was used for further incubation for 24 hours. Meanwhile, DCs were introduced at a rate of 2 × 10⁶ cells per well. 5The densities of DCs were seeded in 1 mL of culture medium into the wells of the lower chamber and co-incubated with 4T1 cells treated in different ways in the upper chamber for 24 h. Subsequently, the DCs were trypsinized and collected by centrifugation, stained with CD86 and CD80 antibodies in the dark for 15 min, respectively. After staining, they were washed with PBS by centrifugation, and finally, the DCs were resuspended in 0.2 mL of PBS for flow cytometry analysis. Figure 19 As shown, Fe 3+ The maturation rates of DCs in the TF (7.93%), Toy (15.9%), and Gos (17.8%) groups were significantly higher than those in the PBS group (1.36%). Furthermore, based on the advantages of combination therapy in inducing ICD in cancer cells, the maturation rates of DCs in the combination therapy groups (TF, GF, and GT) increased to 40.5%, 55.3%, and 66.2%, respectively, and were significantly higher than those in the single-treatment groups (Fe...). 3+ (Toy and Gos). Clearly, due to the significant induction of ICD in cancer cells by GFT NCAs, the maturation rate of DCs in the GFT group was the highest (75.3%). In summary, GFT NCAs effectively promote DC maturation and activate anti-tumor immunity by inducing ICD in 4T1 cells through a dual pathway of inducing mitochondrial dysfunction and exacerbating endoplasmic reticulum stress.

[0135] Example 11

[0136] A 4T1 subcutaneous tumor model was established in 4-5 week old female Balb / c mice. 100 μL of clinical T1 MR contrast agent Magnevist and GFT NCAs solution (60 μg Fe / mouse and 60 μg Gd / mouse, respectively) were injected via tail vein. T1 MR images of the tumor-bearing mice were scanned at different time points (0, 15, 30, 45, 60, 75, 90, and 105 min) after injection using a 3.0T clinical MRI system. The T1 MR signal intensity at the tumor site was measured, and the signal-to-noise ratio (SNR) was calculated to evaluate the in vivo T1 MR contrast effect of the material. Figure 20As shown, at 15 and 30 minutes after tail vein injection, the T1 MR signal at the tumor site in the Magnevist group was stronger than that in the GFT group, reaching a peak at 30 minutes. The T1 MR signal intensity gradually decreased over time, indicating that Magnevist could reach the tumor site more quickly and be metabolized more rapidly. The T1 MR signal intensity in the GFT group gradually increased over time, reaching a peak at 90 minutes, significantly higher than that in the Magnevist group, and decreased at 105 minutes. In conclusion, GFT NCAs can accumulate at the tumor site, exhibiting sustained T1 MR imaging characteristics at the tumor site, with a significantly higher peak T1 MR signal than Magnevist, and are metabolized in vivo over time.

[0137] Example 12

[0138] A 4T1 subcutaneous tumor model was established in female Balb / c mice at 4-5 weeks of age, and the tumor volume was increased to 150 mm². 3 Around 1000 mice, tumor-bearing mice were randomly divided into 4 groups (PBS, free Gos+Fe2+, ... 3+ Mice in the GFT+A-PD-L1 group received tail vein injections of PBS, G+F+T, or GFT NCAs (100 μL, 30 μg Fe / mouse, 30.6 μg Gos / mouse, or 7.44 μg Toy / mouse). Mice in the GFT+A-PD-L1 group received intratumoral injections of A-PD-L1 solution (0.2 mg / mL) one day after GFT NCA injection. -1 (100 μL PBS). The mice were treated four times, once every three days. The tumor volume and body weight of each mouse were recorded every two days for 14 days. The survival status of the mice was recorded every two days for 30 days. The tumor volume and relative tumor volume of the mice were calculated using the following formulas (1) and (2).

[0139] Tumor volume (V) = a × b 2 / twenty one)

[0140] a and b represent the maximum and minimum diameters of the tumor, respectively.

[0141] Relative tumor volume = V / V0 (2)

[0142] V and V0 represent the tumor volume after drug administration and the tumor volume before drug administration, respectively.

[0143] like Figure 21 As shown in Figure A, compared with the PBS group, the free drug group G+F+T showed a certain tumor-suppressive effect, mainly due to the chemotherapeutic effect of the drug and Fe.3+ While CDT mediated tumor suppression, drug accumulation at the tumor site was limited. Compared to the G+F+T group, GFT NCAs showed more significant tumor suppression, mainly due to EPR-mediated enrichment of GFT NCAs at the tumor site. Further combination with ICB therapy resulted in the most significant tumor suppression effect from the GFT NCAs+A-PD-L1 mediated trimodal therapy, and also exhibited the highest survival rate (100%) in mice 30 days after treatment. Figure 21 (B) This further demonstrates the potent antitumor efficacy of the combination therapy of GFT NCAs and A-PD-L1. Conversely, the survival rates of mice in the PBS and G+F+T groups were less than or equal to 60%, significantly lower than those in the GFT+A-PD-L1 group. Furthermore, the stable changes in mouse body weight after different treatments throughout the experiment indicate that the combination therapy of GFT NCAs and A-PD-L1 has good biocompatibility. Figure 21 C).

Claims

1. An iron polyphenol nanocapsule, characterized in that, The nanocapsules are obtained using a microfluidic chip from raw materials containing ferric salts, fentanylmycin, and gossypol. The preparation method of iron polyphenol nanocapsules includes: (1) Mix ferric salt, gossypol, water and ethanol, and sonicate to obtain a mixed solution of gossypol-ferric intermediate; The ratio of the trivalent iron salt, gossypol, water, and ethanol is 0.08~0.15 mg : 0.85~1.25 mg : 10~30 μL : 120.5~145.5 μL; (2) Mix ferric salt, fugamycin, water and ethanol, and sonicate to obtain a fugamycin iron intermediate mixed solution; the ratio of ferric salt, fugamycin, water and ethanol is 0.20~0.40 mg: 0.45~0.65 mg: 3.55~5.55 mL: 0.54~0.75 mL; (3) Using a microfluidic chip, the mixed solution of ferric intermediates of ferric ammonium was injected into the first injection port, the mixed solution of ferric ammonium phenolate was injected into the second injection port, and the buffer solution was injected into the third and fourth injection ports respectively. The solution was collected through the outlet to obtain iron polyphenol nanocapsules. The buffer solution was Tris buffer solution with a pH of 7.4~10.

5. The flow rate ratio of the first, second, third and fourth injection ports was 3~6 : 0.1~0.3 : 35~55 : 3~6.

2. A method for preparing the iron polyphenol nanocapsules according to claim 1, comprising: (1) Mix ferric salt, gossypol, water and ethanol, and sonicate to obtain a mixed solution of gossypol-ferric intermediate; The ratio of the ferric salt, gossypol, water, and ethanol is 0.08~0.15 mg : 0.85~1.25 mg : 10~30 μL : 120.5~145.5 μL; (2) Mix ferric salt, fugamycin, water and ethanol, and sonicate to obtain a fugamycin iron intermediate mixed solution; the ratio of ferric salt, fugamycin, water and ethanol is 0.20~0.40 mg: 0.45~0.65 mg: 3.55~5.55 mL: 0.54~0.75 mL; (3) Using a microfluidic chip, the mixed solution of fulvamycin iron intermediates was injected into the first injection port, the mixed solution of gossypol iron intermediates was injected into the second injection port, and the buffer solution was injected into the third and fourth injection ports respectively. The iron polyphenol nanocapsules were collected through the outlet. The buffer solution was Tris buffer solution with a pH of 7.4~10.

5. The flow rate ratio of the first, second, third and fourth injection ports was 3~6 : 0.1~0.3 : 35~55 : 3~6.

3. The preparation method according to claim 2, characterized in that, In step (1), the ferric salt is anhydrous ferric chloride; the ultrasonic time is 1~5 min.

4. The preparation method according to claim 2, characterized in that, In step (2), the ferric salt is anhydrous ferric chloride; the ultrasonic time is 1~5 min.

5. The preparation method according to claim 2, characterized in that, The microfluidic chip in step (3) includes: a first inlet, a second inlet, a third inlet, a fourth inlet, an S-shaped microfluidic channel, and an outlet connected in sequence; wherein the first inlet, the second inlet, the third inlet, the fourth inlet, the S-shaped microfluidic channel, and the outlet are connected through the microfluidic channel, and the third inlet includes a third sub-inlet 1 and a third sub-inlet 2, which are located on both sides of the microfluidic channel.

6. The preparation method according to claim 5, characterized in that, The height of each microfluidic channel is 30~100 μm, the width of the microfluidic channels of the first, second and third inlets is 80~150 μm, the width of the remaining microfluidic channels is 200~400 μm, and the total length from the channel inlet to the outlet is 30.5~38.5 mm.

7. The use of the iron polyphenol nanocapsule of claim 1 in the preparation of antitumor drugs or T1 magnetic resonance imaging contrast agents.

8. The use of the iron polyphenol nanocapsule of claim 1 in combination with PD-L1 antibody in the preparation of an antitumor drug.

Images