A nano-assembly for enhancing photodynamic-immunotherapy and a preparation method and application thereof

Abstract

The application discloses a kind of nano-assemblies of enhanced photodynamic-immunotherapy and its preparation method and application, belong to the technical field of pharmaceutical preparation.The nano-assemblies are co-assembled by photodynamic photosensitizer and cystine / glutamate antiporter inhibitor through intermolecular force of π-π stacking and hydrophobic interaction, and are surface-modified with amphiphilic PEG modifier, the molar ratio of the photodynamic photosensitizer and cystine / glutamate antiporter inhibitor is 10:1~1:10, and the mass ratio of the total mass of the photodynamic photosensitizer and cystine / glutamate antiporter inhibitor and the mass of PEG modifier is 60:40~95:5.The preparation method of the nano-assemblies of the application is simple and convenient, the drug loading efficiency is high, the efficient co-loading of drug can be realized, and the effect of enhancing photodynamic-immunotherapy is achieved, so the nano-assemblies have good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, specifically relating to a nanoassembly based on glutathione downregulation to enhance photodynamic-immunotherapy, its preparation method, and its application. Background Technology

[0002] Immunotherapy has emerged as a revolutionary cancer treatment method, demonstrating immense potential in leveraging the body's immune system to fight tumors. While immunotherapy promises to eliminate existing tumors and prevent recurrence, it's important to note that current immunotherapies are only beneficial to specific cancer patient populations. This limitation stems from the fact that tumor cells often exhibit low immunogenicity, evading detection by immune cells such as dendritic cells and T lymphocytes. Therefore, evoking an effective anti-tumor immune response at the tumor site remains a significant challenge.

[0003] Some cancer therapies, including chemotherapy, photodynamic therapy (PDT), and radiotherapy, not only eliminate tumor cells but also trigger a powerful immune response. PDT is particularly noteworthy in cancer treatment due to its unique tumor selectivity, non-invasiveness, and minimal systemic toxicity. Under laser irradiation, excited photosensitizers transfer their energy to molecular oxygen, leading to the generation of reactive oxygen species (ROS). These ROS initiate damage to key intracellular targets at the organelle and molecular levels. This process not only directly eliminates tumor cells but also stimulates the release of damage-associated molecular patterns (DAMPs), such as calreticulin (CRT), high-mobility group protein B1 (HMGB1), and adenosine triphosphate (ATP). Furthermore, DAMPs promote dendritic cell maturation, facilitate antigen presentation, and trigger anti-tumor immune responses. However, in ROS-induced intracellular photodamage, the essential intracellular antioxidant glutathione (GSH) counteracts oxidative stress to maintain redox homeostasis. This defense mechanism weakens the efficacy of PDT.

[0004] Existing research indicates that glutathione plays a crucial role in maintaining intracellular redox homeostasis. Certain iron-lowering agents, such as Erastin, act by inhibiting the cysteine / glutamate antitransporter, leading to downregulation of GSH expression. This disruption of the intracellular antioxidant system interferes with redox balance. However, to date, no research has been reported on the preparation of nanoassemblies using cysteine / glutamate antitransporter inhibitors and photodynamic photosensitizers for synergistic antitumor effects. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention aims to provide a nano-assembly based on glutathione downregulation to enhance photodynamic-immunotherapy, its preparation method, and its application. The present invention constructs a hybrid nano-assembly based on glutathione downregulation to enhance photodynamic-immunotherapy using molecular hybrid nanoassembly technology. Preferably, the photodynamic photosensitizer pyrophyllofoetate a (PPa) and the cystine / glutamate antitransporter inhibitor Erastin (Era) are constructed using a one-step nanoassembly method followed by DSPE-PEG. 2k After modification, the nanoparticles were co-assembled into nanoassemblies (PPa@Era NAs). The prepared nanoparticles exhibited uniform particle size (around 120 nm), a PDI of less than 0.2, a zeta potential of around -20 mV, and good stability. Under 660 nm laser irradiation, PPa nanoparticles immediately generated a large amount of singlet oxygen, while Era inhibited the cysteine / glutamate antitransporter, limiting intracellular glutathione synthesis, preventing the detoxification effect of tumor cells, and further promoting immunogenic cell death in tumor cells. This resulted in more efficient photodynamic-immunotherapy, solving the technical problem of limited efficacy of existing photodynamic-immunotherapy in anti-tumor processes.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a nano-assembly for enhancing photodynamic-immunotherapy, wherein the nano-assembly is co-assembled from a photodynamic photosensitizer and a cysteine / glutamate antitransporter inhibitor through π-π stacking and hydrophobic intermolecular forces, and is modified with a PEG modifier. The molar ratio of the photodynamic photosensitizer and the cysteine / glutamate antitransporter inhibitor is 10:1 to 1:10, and the mass ratio of the total mass of the photodynamic photosensitizer and the cysteine / glutamate antitransporter inhibitor to the mass of the PEG modifier is 60:40 to 95:5.

[0008] Preferably, the molar ratio of the photodynamic photosensitizer and the cystine / glutamate antitransporter inhibitor is 3:1.

[0009] Furthermore, the intermolecular forces include π-π stacking, hydrophobic interactions, and hydrogen bonds.

[0010] Furthermore, the photodynamic photosensitizer includes one or more of chlorophyll a, pheophytin a, pyropheophytin a, pyropheophytin a hexyl ether, and dihydroporphyrin E6.

[0011] Preferably, the photodynamic photosensitizer is pyrophyllin a.

[0012] Furthermore, the cystine / glutamate antitransporter inhibitor includes one or more of Erastin, glutamate, sorafenib, and sulfasalazine.

[0013] Preferably, the cystine / glutamate reverse transporter inhibitor is Erastin.

[0014] Furthermore, the PEG modifier includes one or more of PCL-PEG, DSPE-PEG, DSPE-SS-PEG, PLGA-PEG, or PE-PEG, and the molecular weight of the PEG is 200-20000.

[0015] Preferably, the PEG modifier is DSPE-PEG. 2K .

[0016] Secondly, the present invention provides a method for preparing the nano-assembly for enhanced photodynamic-immunotherapy, comprising the following steps:

[0017] The photodynamic photosensitizer, cystine / glutamate antitransporter inhibitor, and PEG modifier were dissolved in organic solvents and mixed thoroughly. The resulting mixed solution was then slowly added dropwise to water while stirring, spontaneously forming uniform co-assembled nanoparticles. The organic solvent was then removed to obtain the final product.

[0018] Furthermore, the organic solvent is one or any combination of two of tetrahydrofuran, dimethyl sulfoxide, and anhydrous ethanol.

[0019] Preferably, the organic solvent is a mixed solution of anhydrous ethanol and tetrahydrofuran in a volume ratio of 1:1.

[0020] Furthermore, methods for removing organic solvents include solvent evaporation, ultrafiltration, or membrane permeation.

[0021] Preferably, the organic solvent removal method is a solvent evaporation method.

[0022] Thirdly, the present invention provides the application of the aforementioned enhanced photodynamic-immunotherapy nanoassemblies in the preparation of drug delivery systems.

[0023] Fourthly, the present invention provides the application of the aforementioned enhanced photodynamic-immunotherapy nanoassemblies in the preparation of antitumor drugs.

[0024] Fifthly, the present invention provides the application of the aforementioned enhanced photodynamic-immunotherapy nanoassemblies in the preparation of injection, oral, or topical drug delivery systems.

[0025] The advantages of this invention over the prior art are as follows:

[0026] 1. This invention prepares a hybrid nanoassembly of a photodynamic photosensitizer (preferably PPa) and a cysteine / glutamate antitransporter inhibitor (preferably Erastin) to enhance photodynamic-immunotherapy for cancer. Under laser irradiation, the singlet oxygen generated by PPa can not only directly kill cells and exert photodynamic therapy, but also promote immunogenic cell death in tumor cells and activate the immune process. Erastin can inhibit the cysteine / glutamate antitransporter, blocking the raw materials for glutathione synthesis, thereby downregulating the intracellular glutathione level and enhancing the photodynamic-immunotherapy effect of PPa.

[0027] 2. The photodynamic photosensitizer (preferably PPa) and cystine / glutamate antitransporter inhibitor (preferably Erastin) hybrid nanoassemblies prepared by this invention have high drug loading (80%) and good colloidal stability, which meet the urgent clinical demand for novel nano-formulations and provide a new strategy for the synergistic use of photosensitizers and other drugs in photodynamic therapy, significantly enhancing the effect of photodynamic therapy. Attached Figure Description

[0028] Figure 1 The diagram shows the synergistic cytotoxicity results of mixed solutions of PPa and Era at different molar ratios in Example 1 of the present invention. In the diagram, A: cytotoxicity results of PPa solution; B: cytotoxicity results of Era solution; C: results of mixed solutions of PPa and Era at different ratios.

[0029] Figure 2 The Malvern particle size distribution and appearance diagram of the non-PPa@Era NAs in Example 2 of the present invention are shown.

[0030] Figure 3 The images show the Malvern particle size distribution, appearance, and transmission electron microscopy (TEM) images of PPa@Era NAs from Example 2 of this invention.

[0031] Figure 4 This is a colloidal stability diagram of PPa@Era NAs in Example 3 of the present invention.

[0032] Figure 5 The figure shows the molecular docking simulation results of PPa@Era NAs in Example 4 of the present invention.

[0033] Figure 6 The figure shows the results of the molecular force disruption experiment of PPa@Era NAs in Example 4 of the present invention.

[0034] Figure 7 This is the ultraviolet absorption spectrum of PPa@Era NAs in Example 4 of the present invention.

[0035] Figure 8These are confocal microscopy images of cellular uptake of PPa@Era NAs at 1 hour (A) and 4 hours (B) in Example 5 of the present invention.

[0036] Figure 9 The flow cytometry plots show the 1-hour and 4-hour cellular uptake of PPa@Era NAs in Example 5 of this invention.

[0037] Figure 10 This is a diagram showing the cytotoxicity results of PPa@Era NAs on 4T1 cells in Example 6 of the present invention.

[0038] Figure 11 This is a diagram showing the cytotoxicity results of PPa@Era NAs on CT26 cells in Example 6 of the present invention.

[0039] Figure 12 This is a blood concentration-time curve of PPa@Era NAs in Example 7 of the present invention.

[0040] Figure 13 This shows the in vitro tissue distribution of PPa@Era NAs in Example 8 of the present invention.

[0041] Figure 14 This is a mouse tumor growth curve from an in vivo anti-tumor experiment in Example 9 of the present invention.

[0042] Figure 15 This is a graph showing the tumor weight in mice during the in vivo anti-tumor experiment of Example 9 of the present invention.

[0043] Figure 16 This is an H&E staining image of the main organs and tumors of mice in the in vivo anti-tumor experiment of Example 9 of the present invention.

[0044] Figure 17 This is a graph showing the change in body weight of mice in an in vivo anti-tumor experiment according to Example 9 of the present invention.

[0045] Figure 18 The results are the liver and kidney function analysis results of mice in Example 9 of this invention.

[0046] Figure 19 This is a diagram showing the maturation analysis of DC cells in the tumor-draining lymph nodes in Example 10 of the present invention.

[0047] Figure 20 This is a diagram showing the maturation analysis of CD4+ T lymphocytes infiltrating the tumor in Example 10 of the present invention.

[0048] Figure 21 This is a diagram showing the maturation analysis of tumor-infiltrating CD8+ T cells in Example 10 of the present invention.

[0049] Figure 22This is a graph showing the serum IFN-γ level in Example 10 of the present invention.

[0050] Figure 23 This is a graph showing the serum TNF-α level in Example 10 of the present invention. Detailed Implementation

[0051] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0052] To better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below. It should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a clearer and more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0053] Example 1: Screening for the optimal synergistic ratio of PPa and Erastin (Era)

[0054] Using 4T1 cells as a cell model, the MTT assay was employed to investigate the synergistic cytotoxicity of PPa and Era. Healthy cells were digested and diluted with culture medium to a concentration of 1 × 10⁻⁶. 4 Cells were prepared at a density of 200 μL / well (2000 cells / well) and mixed by pipetting. The cells were then incubated for 12 hours. Next, serially diluted PPa, Era, and PPa / Era mixtures were added to the 96-well plates, and incubated for another 4 hours. Finally, the cells were exposed to a laser (660 nm, 50 mW / cm²). 2 After incubating for 5 minutes, remove the 96-well plate and add 5 mg mL to each well. -1 Add 20 μL of MTT solution to each well and incubate for 4 hours. After incubation, spin the plate and invert it onto filter paper to thoroughly absorb any remaining liquid. Add 200 μL of DMSO to each well and shake for 10 minutes to dissolve the blue-purple crystals. Measure the absorbance of each well at 490 nm using a microplate reader after zeroing.

[0055] The results are as follows Figure 1 As shown in Table 1, the results indicate that the mixed solution with a molar ratio of PPa to Era of 3:1 (PPa / Era) exhibits the highest cytotoxicity, demonstrating the best synergistic effect, with a combination index (CI) value of 0.49.

[0056] Table 1. Synergistic Index (CI) of PPa / Era Mixed Solutions with Different Molar Ratios

[0057]

[0058] Example 2: Preparation of PPa@Era NAs

[0059] (1) Preparation method of non-PEGylated PPa@Era NAs: Accurately weigh 5 mg of PPa and 5 mg of Era, and dissolve them separately in 1 mL of a mixed organic solvent (anhydrous ethanol / tetrahydrofuran (V / V) = 1:1). Add the mixture of PPa solution (75 μL) and Era solution (25 μL) dropwise to deionized water (2 mL) under magnetic stirring. Remove the organic solvent from the nano-assembly system under vacuum at 30 °C.

[0060] (2)DSPE-PEG 2K Preparation method of modified nanoparticles: Accurately weigh 5 mg of PPa, 5 mg of Era, and DSPE-PEG. 2k 10 mg was dissolved in 1 mL of a mixed organic solvent (anhydrous ethanol / tetrahydrofuran (V / V) = 1:1). The PPa solution (75 μL), Era solution (120 μL), and DSPE-PEG were then dissolved. 2k A mixture of 25 μL was added dropwise to 2 mL of deionized water under magnetic stirring. The organic solvent in the nano-assembly system was removed under vacuum at 30 °C. The particle size and particle size distribution of the prepared nanoparticles were determined by dynamic light scattering.

[0061] The results are as follows Figure 2 As shown, the average particle size of non-PPa@Era NAs is approximately 95 nm, indicating good nano-assembly capabilities. Furthermore, the average diameter of PEGylated PPa@Era NAs slightly increased to approximately 120 nm, and their Zeta potential decreased from approximately -12 mV to -24 mV, thereby improving colloidal stability. Figure 3 (See Table 2). As expected, PEGylation significantly improved the stability of PPa@Era NAs. Furthermore, transmission electron microscopy (TEM) showed that PPa@Era NAs were uniformly spherical.

[0062] Table 2. Particle size, particle size distribution, and zeta potential of non-PPa@Era NAs and PEGylated PPa@Era NAs nanoparticles

[0063]

[0064] Example 3: Colloidal stability experiment of PEGylated PPa@Era NAs

[0065] The PPa@Era NAs (1 μmol mL) prepared in Example 2 were used. -1The sample was added to PBS (pH 7.4) containing 10% FBS and incubated in a shaker at 37°C for 24 hours. Particle size changes were measured by dynamic light scattering at predetermined time points (0, 2, 4, 8, 12, and 24 hours). Results are as follows: Figure 4 As shown, the particle size did not change significantly within 24 hours, indicating that PPa@Era NAs have good colloidal stability.

[0066] Example 4: Assembly Mechanism Analysis of PPa and Era

[0067] The molecular interaction forces between PPa and Era were investigated using molecular docking simulation technology on the YInfotek cloud computing platform (http: / / cloud.yinfotek.com / ), exploring the assembly mechanism driving the nanoparticles. The chemical structures of PPa and Era were plotted using Chemdraw 17.1 software, then converted to 3D structures. Energy minimization was then performed under an MFF94 force field, and finally, semi-flexible docking was performed using the AutoDock Vina program to output the optimal conformation and corresponding assembly forces.

[0068] Simultaneously, intermolecular force disruption experiments were conducted, involving the addition of PPa@Era NAs to solutions of potassium chloride (KCl, 10 mM), sodium dodecyl sulfate (SDS, 10 mM), or urea (Urea, 10 mM), followed by incubation at 37°C in a shaker. The particle size of PPa@Era NAs was characterized using a Malvern Zetasizer at predetermined time points. The assembly mechanism of the nanoparticles was further investigated using UV spectroscopy. The UV absorption spectra of PPa solution (5 μg / mL) and PPa@Era NAs (5 μg / mL, PPa equivalent) were measured using a microplate reader.

[0069] Molecular docking simulation results show that the intermolecular forces between PPa and Era are mainly hydrophobic interactions and π-π stacking forces. Figure 5 Subsequently, the assembly mechanism was further investigated through intermolecular force disruption experiments. Figure 6 As shown, the particle size of PPa@Era NAs did not change significantly in KCl and Urea solutions. In contrast, the particle size of PPa@Era NAs increased significantly in SDS solution, indicating that hydrophobic forces play a crucial role in the assembly process of PPa@Era NAs. The UV absorption spectrum of PPa@Era NAs showed a significant red shift compared to the PPa solution, proving the presence of π-π stacking forces within them. Figure 7 Therefore, the assembly process of PPa@EraNAs is mainly driven by π-π stacking and hydrophobic interactions, which is consistent with the results of molecular docking simulations.

[0070] Example 5: Cellular uptake of PPa@Era NAs

[0071] After normal digestion, healthy 4T1 cells were centrifuged at low speed (1000 rpm / min, 3 min) to remove trypsin. A cell suspension of a specific concentration was prepared by adding freshly prepared RPMI 1640 medium. The cell suspension was then thoroughly mixed and added to 1 mL of cell suspension (50,000 cells / well) per well of a 24-well plate containing cell spreaders. The plate was then incubated in a cell culture incubator (37℃, 5% CO2) for 12 h. PPA solution and PPA@Era NAs were diluted with fresh blank RPMI 1640 medium to achieve an equivalent PPA concentration of 2.5 μg / mL. 1 mL of the drug-containing medium was added to each well, and the plates were then incubated for 1 h and 4 h. After the specified time, the cells were washed, fixed, and finally analyzed using a confocal microscope to assess cell uptake.

[0072] Cell uptake was then quantitatively observed using flow cytometry, with the following procedure: Cell plating was performed as above in 12-well plates (200,000 cells / well). Drug administration was also performed as above. After drug administration and incubation, the cell culture plate was removed, the drug-containing medium was discarded, and cold PBS was immediately added to stop cell uptake. Cell digestion was then performed, and the cell suspension was transferred to a labeled 1.5 mL EP tube. After centrifugation at low temperature (4°C, 1000 rpm) for 3 min, the supernatant was discarded, and the cells were resuspended in 500 μL PBS (pH 7.4). The cells were then filtered through a 70 μm cell filter and added to flow cytometry tubes for cell uptake measurement.

[0073] The above experimental results indicate that cellular uptake of both PPa solution and PPa@Era NAs is time-dependent. In contrast, the cellular uptake efficiency of PPa@Era NAs at 1 h and 4 h was significantly higher than that of PPa solution. Figure 8 and Figure 9 ).

[0074] Example 6: Cytotoxicity of PPa@Era NAs

[0075] Using 4T1 and CT26 cells as cell models, the cytotoxicity of PPa@Era NAs was evaluated using the MTT assay. Cells (2 × 10⁶ cells) were... 3 Cells were seeded in 96-well plates for 12 h. Then, the old medium was replaced with fresh medium containing concentration gradients of Era solution, PPa solution, PPa / Era mixed solution, and PPa@Era NAs. After 4 h of incubation, the light group was exposed to a 660 nm laser (50 mW cm⁻¹). 2After culturing the cells in the dark for 44 hours (5 min), remove the cells from the 96-well plate and add 5 mg mL to each well. -1 Add 20 μL of MTT solution to each well and incubate for 4 hours. After incubation, spin the plate and invert it onto filter paper to thoroughly absorb any remaining liquid. Add 200 μL of DMSO to each well and shake for 10 minutes to dissolve the blue-purple crystals. Measure the absorbance of each well at 490 nm using a microplate reader after zeroing.

[0076] The results of the above cytotoxicity experiments are as follows: Figure 10 and Figure 11 As shown, PPa@Era NAs exhibited the best cytotoxicity (PPa@Era NAs+L) under laser irradiation compared to other formulation groups, indicating that the synergistic delivery of PPa and Era has a significant advantage in enhancing cytotoxicity.

[0077] Example 7: Pharmacokinetic Study of PPa@Era NAs

[0078] SD rats weighing 180-220g were randomly divided into groups and fasted for 12 hours before administration, but allowed free access to water. PPa solution and PPa@Era NAs prepared in Example 2 (both 1.5 mg / kg based on PPa) were administered intravenously. Blood samples were collected from the orbital sinus at specified time points (0.033, 0.083, 0.25, 0.5, 1, 2, 4, 8, and 12 hours), and plasma was separated (13000 rpm, 5 min). PPa was then extracted by centrifugation and protein precipitation. Finally, the pharmacokinetic behavior of each formulation was detected using an ELISA reader (excitation 415 nm, emission 675 nm). The experimental results are as follows: Figure 12 As shown in Table 3, PPa solution is rapidly metabolized and cleared due to its short half-life. Compared to the solution, PPa@Era NAs exhibit a significantly prolonged cycle time and a markedly improved AUC. This is attributed to its excellent assembly ability, superior colloidal stability, and the PEG-modified hydrophilic layer on its surface, which provides a favorable foundation for drug accumulation in tumors in vivo.

[0079] Table 3. Pharmacokinetic parameters of PPa solution and PPa@Era NAs (n=5)

[0080]

[0081] a) Area under the plasma concentration-time curve to infinity (μg / mL*h); b) Half-life (h); c) Mean residence time (h).

[0082] Example 8: In vitro distribution experiment of PPa@Era NAs

[0083] 4T1 cell suspension was inoculated into BALB / c mice. When the tumor volume reached 400 mm², the cells were cultured. 3 Mice were administered PPa solution and PPa@Era NAs (both calculated as 1.5 mg / kg PPa) via tail vein injection. Mice were euthanized at 2, 4, and 8 hours post-administration for fluorescence intensity analysis of isolated tissues and organs.

[0084] The results are as follows Figure 13 As shown, the highest tumor accumulation was observed in both PPa solution and PPa@Era NAs at 2 hours. In contrast, the fluorescence intensity of PPa@Era NAs at the tumor site was significantly higher at 2, 4, and 8 hours than that of the PPa solution group, which may be related to the longer blood circulation time and EPR effect of PPa@Era NAs.

[0085] Example 9: In vivo antitumor experiment of PPa@Era NAs

[0086] The in vivo antitumor activity of PPa@Era NAs was studied using 4T1 tumor-bearing mice (20-22g). First, 100μL of 4T1 cells (5×10⁻⁶ g) were... 7 (cells / mL) were subcutaneously injected into the right back of mice. When the tumor volume reached 150 mm², the tumor was treated. 3 Around 10:00 AM, tumor-bearing mice were randomly divided into 6 groups (n=5): PBS group, Era solution group, PPa@Era NAs group, PPa solution + light irradiation group, PPa / Era mixed solution + light irradiation group, and PPa@Era NAs + light irradiation group (all 2 mg / kg based on PPa). The mice were treated with the medication every other day for 5 treatments. The light irradiation group received laser irradiation (660 nm, 50 mW / cm²) 2 hours after injection. 2 (5 min), mouse body weight and tumor volume were measured and recorded daily during the treatment period. After the last treatment, the mice were sacrificed, and blood samples were collected for liver and kidney function analysis. Heart, liver, spleen, lung, kidney, and tumor were collected for H&E staining, and the excised tumors were weighed.

[0087] The results are as follows Figure 14-15 As shown, the solution groups (Era solution, PPa solution + light irradiation, PPa / Era solution + light irradiation) only exhibited moderate tumor-suppressive effects, attributed to their rapid clearance from the bloodstream and insufficient tumor accumulation. Notably, under laser irradiation, PPa@Era NAs significantly eliminated tumors, demonstrating a clear advantage over other formulations. H&E staining results showed that after treatment with PPa@Era NAs under laser irradiation, extensive tumor apoptosis and necrosis occurred. Figure 16The potent therapeutic effects of PPa@Era NAs are attributed to their excellent assembly ability, good colloidal stability, efficient cellular uptake, synergistic cytotoxicity, superior pharmacokinetics, and tumor-specific accumulation. Furthermore, no significant changes were observed in mouse body weight and hematological parameters. Figure 17 and Figure 18 This indicates that PPa@Era NAs have good biological safety.

[0088] Example 10: In vivo immune activation

[0089] After the efficacy experiment was completed, tumor draining lymph nodes, tumor tissue and serum were collected. Immunofluorescence staining and ELISA kits were used to determine the maturation of dendritic cells, the infiltration of CD4+ T lymphocytes and CD8+ T lymphocytes in the tumor site, and the levels of immune factors (including TNF-α and IFN-γ) in the serum.

[0090] The results are as follows Figure 19-23 As shown, under 660nm laser irradiation, the PPa@EraNAs treatment group exhibited the highest proportion of mature dendritic cells (DCs) and CD4+ and CD8+ T lymphocyte infiltration. Furthermore, immunogenicity assays demonstrated that the PPa@EraNAs treatment group showed the highest levels of TNF-α and IFN-γ in the mouse serum, indicating that the nanoassemblies elicited the strongest immune response.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nanoassembly for enhancing photodynamic-immunotherapy, characterized in that, The nano-assembly is co-assembled from a photodynamic photosensitizer and a cystine / glutamate antitransporter inhibitor through intermolecular forces and modified with a PEG modifier. The molar ratio of the photodynamic photosensitizer and the cystine / glutamate antitransporter inhibitor is 10:1 to 1:10, and the mass ratio of the total mass of the photodynamic photosensitizer and the cystine / glutamate antitransporter inhibitor to the PEG modifier is 60:40 to 95:

1. The photodynamic photosensitizer is pyrophylloxera a; The cystine / glutamate antitransporter inhibitor is Erastin; The PEG modifier is DSPE-PEG. 2K ; The method for preparing the nanoassemblies for enhanced photodynamic-immunotherapy includes the following steps: The photodynamic photosensitizer, cystine / glutamate antitransporter inhibitor, and PEG modifier were dissolved in organic solvents and mixed thoroughly. The resulting mixed solution was then slowly added dropwise to water under stirring, spontaneously forming uniform co-assembled nanoparticles. The organic solvent was then removed to obtain the final product.

2. The nanoassembly for enhanced photodynamic-immunotherapy as described in claim 1, characterized in that, The organic solvent is one or any combination of two of anhydrous ethanol, tetrahydrofuran, and dimethyl sulfoxide.

3. The use of the nanoassemblies for enhanced photodynamic-immunotherapy as described in any one of claims 1-2 in the preparation of drug delivery systems.

4. The use of the enhanced photodynamic-immunotherapy nanoassembly according to any one of claims 1-2 in the preparation of antitumor drugs.

5. The use of the enhanced photodynamic-immunotherapy nanoassembly according to any one of claims 1-2 in the preparation of injection, oral or topical drug delivery systems.

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