Ion pair engineered nanoassemblies inducing type i and ii immunogenic cell death and construction and use thereof

Abstract

The application belongs to the field of new adjuvants and new dosage forms of pharmaceutical preparations, and particularly relates to a type I and type II immunogenic cell death inducer ion pairing engineered nanoassemblies as well as construction and application thereof. The nanoassemblies are composed of type I ICD inducers, type II ICD inducers, hydrophobic auxiliary counterions and amphiphilic lipid ion pairing. The type I ICD inducer is selected from mitoxantrone, doxorubicin, oxaliplatin or cyclophosphamide; the type II ICD inducer is selected from hypericin; and the hydrophobic auxiliary counterion is selected from cholesteryl sodium sulfate, cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, glycolcholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, deoxycholic acid lysine derivative, oleanolic acid and ursolic acid. The type I and type II ICD inducers are combined in the application, and have a synergistic tumor treatment effect.

Description

Technical Field

[0001] This invention belongs to the field of new excipients and dosage forms for pharmaceutical preparations, specifically the construction of type I and type II immunogenic cell death inducing agent ion-paired engineered nanoassemblies, and the construction of such assemblies. In particular, it relates to the construction of a chemotherapeutic drug that induces immunogenic cell death and a photosensitizer ion-paired engineered nanoassembly that induces immunogenic cell death, and the synergistic antitumor effect provided by combination chemotherapy, phototherapy and immunotherapy. Background Technology

[0002] Malignant tumors pose a serious threat to human health. Traditional treatments (surgery, chemotherapy, and radiotherapy) are highly invasive, have severe side effects, and are prone to multidrug resistance, failing to effectively inhibit tumor recurrence and metastasis. In recent years, novel anti-tumor strategies, represented by immunotherapy, have developed rapidly, showing promising clinical application prospects. However, immunotherapy is only effective for a small percentage of tumor patients with immunogenic characteristics (≈10-30%). Therefore, improving the immunogenicity of tumors is crucial to enhancing anti-tumor immunotherapy.

[0003] Immunogenicity depends on antigenicity and adjuvant properties. Inducing immunogenic cell death (ICD) in tumor cells through chemotherapy, radiotherapy, and phototherapy generates tumor antigens through cytotoxicity and releases damage-associated molecular patterns (DAMPs) to act as adjuvants, thereby stimulating a specific immune response and inhibiting tumor growth, metastasis, and recurrence. ICD inducers can be classified into type I and type II based on their DAMP-inducing mechanisms. Type I ICD inducers act directly on cytoplasmic proteins or cell membrane channels, or indirectly on the endoplasmic reticulum (ER) through a series of metabolic pathways. Type II ICD inducers act directly on the ER, inducing an ER stress response by altering ER homeostasis. Current research mainly focuses on using single-type ICD inducers, which often have limited effects and are frequently used in combination with other immunotherapies. Combining type I and type II ICD inducers holds promise for synergistically killing tumor cells to produce antigens and promoting DAMP release through multiple mechanisms, thereby enhancing the immune system's recognition of tumor antigens. Furthermore, the combined use of type I and type II ICD inducers holds promise for achieving highly efficient synergy across different treatment modalities (chemotherapy-phototherapy-immunotherapy). However, the significant differences in the physicochemical properties of type I and type II ICD inducers pose a challenge to their co-loading into a single drug delivery system. Therefore, there is an urgent need to develop a drug delivery platform for the co-loading of type I and type II ICD inducers.

[0004] In recent years, the widespread application of small molecule self-assembled nanodelivery systems in the biomedical field has greatly enriched drug delivery and tumor treatment strategies. Based on the structural characteristics of some drugs containing ionizable groups, this invention proposes a novel strategy combining hydrophobic ion pairing with self-assembled nanodelivery systems. This method utilizes hydrophobic counterions to form ion pairs with ionizable drugs through electrostatic interactions, achieving self-assembly by balancing the interactions between the two. These ion-paired engineered nanoassemblies enable the co-delivery of drugs with different physicochemical properties, achieving efficient co-loading of type I and type II ICD inducers. Summary of the Invention

[0005] This invention designs and constructs engineered nanoassemblies with ion-paired structure of type I and type II ICD inducers, exhibiting synergistic effects in inducing immunogenic cell death, particularly the hypericin-mitoxantrone ion-paired engineered nanoassembly. This achieves efficient co-loading and co-delivery of the chemotherapeutic drug mitoxantrone (MTO) and the photosensitizer hypericin (HY) through non-covalent interactions. The ion-paired engineered nanoassemblies of this invention can exert a highly efficient synergistic anti-tumor effect through chemotherapy combined with phototherapy. Simultaneously, the chemotherapeutic and phototherapy drugs can synergistically induce immunogenic cell death through different mechanisms, enhancing anti-tumor immunity. This invention provides a novel, safe, and effective drug delivery strategy for combination therapy.

[0006] The present invention achieves the above objectives through the following technical solutions:

[0007] The first aspect of the present invention provides a nano-assembly for synergistic anti-tumor effects of chemotherapy, phototherapy and immunotherapy, wherein the nano-assembly is composed of a type I ICD inducer, a type II ICD inducer, a hydrophobic auxiliary counterion and an amphiphilic lipid ion pair;

[0008] The type I ICD inducer is selected from mitoxantrone, doxorubicin, oxaliplatin, or cyclophosphamide; the type II ICD inducer is selected from hypericin; the hydrophobic auxiliary counterion is selected from sodium cholesterol sulfate (SCS), cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, deoxycholic acid lysine derivative, oleanolic acid, and ursolic acid.

[0009] In the above technical solution, the amphiphilic lipid is further selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, and methoxy polyethylene glycol 2000-distearate phosphatidylethanolamine (DSPE-PEG). 2K Or cholesterol-polyethylene glycol 2000 (CH-PEG).

[0010] In the above technical solution, the molar ratio of type I ICD inducer, type II ICD inducer, hydrophobic auxiliary counterion, and amphiphilic lipid is 1:(0.1-10):(1-10):(0.1-1).

[0011] In the above technical solution, the type I ICD inducer is selected from mitoxantrone, and the type II ICD inducer is selected from hypericin.

[0012] The type I and type II ICD inducer ion pairing engineered nanoassemblies described in this invention are nanoassemblies formed by type I and type II ICD inducers through non-covalent forces, such as electrostatic interactions, hydrophobic interactions, and intermolecular hydrogen bonds.

[0013] Specifically, an ionizable drug is used to form an ion pair with an auxiliary counterion. This ion pairing strategy shields the inherent charge of the drug molecule and expands the hydrophobic region of the drug molecule, thereby improving the physicochemical properties of the drug molecule. Finally, self-assembly is achieved by utilizing the balance of interactions between the drug molecule and the auxiliary molecule.

[0014] A second aspect of this invention provides a method for preparing the aforementioned type I and type II ICD inducer ion-pairing engineered nanoassemblies, comprising the following steps:

[0015] a. Dissolve type I ICD inducer, type II ICD inducer, hydrophobic auxiliary counterion and amphiphilic lipid in dimethyl sulfoxide (DMSO);

[0016] b. Under ultrasound probe, the solution obtained in step a is slowly added dropwise to water to form a uniform nanocolloid solution, namely, the type I and type II ICD inducer ion pairing engineered nanoassemblies.

[0017] In the above technical solution, further, in step b, the ultrasonic power is 100-400W and the ultrasonic time is 1-15min.

[0018] In the above technical solution, further, in step b, the volume of water is 8-20 times the volume of dimethyl sulfoxide (DMSO) in step a.

[0019] In the above technical solution, further, the molar ratio of type I ICD inducer: type II ICD inducer: hydrophobic auxiliary counterion: amphiphilic lipid in step a is 1:(0.1-10):(1-10):(0.1-1);

[0020] Furthermore, the molar ratio of type ICD inducer: type II ICD inducer: hydrophobic auxiliary counterion: amphiphilic lipid can be 9:1:50:2, 7:1:40:1.6, 5:1:30:1.2, 3:1:20:0.8, 1:1:10:0.4, 1:3:20:0.8, 1:5:30:1.2, 1:7:40:1.6, 1:9:50:2;

[0021] When the molar ratio of type ICD inducer: type II ICD inducer: hydrophobic auxiliary counterion: amphiphilic lipid is 9:1:50:2, 7:1:40:1.6, 5:1:30:1.2, 3:1:20:0.8, 1:1:10:0.4, 1:3:20:0.8, 1:5:30:1.2, 1:7:40:1.6, or 1:9:50:2, better co-assembled nanoparticles can be formed.

[0022] In the above technical solution, the preferred type I and type II ICD inducer ion-pairing engineered nanoassemblies of the present invention are hypericin-mitoxantrone ion-pairing engineered nanoassemblies. The hypericin-mitoxantrone ion-pairing engineered nanoassemblies described in step b above have a particle size of 80–200 nm, uniform particle size distribution, and drug loadings of mitoxantrone and hypericin of 5–30%, respectively.

[0023] The hypericin-mitoxantrone ion-paired engineered nanoassemblies are formed by mitoxantrone and the hydrophobic counterion sodium cholesterol sulfate, which shields the intrinsic charge of mitoxantrone and expands the hydrophobic region of mitoxantrone. Finally, the nanoassemblies are formed by the balance of non-covalent forces between mitoxantrone, hypericin and the auxiliary molecule sodium cholesterol sulfate, such as electrostatic interaction, hydrophobic interaction and intermolecular hydrogen bonding.

[0024] Among them: mitoxantrone: hyperoside: sodium cholesterol sulfate: DSPE-PEG 2K The molar ratios are 9:1:50:2, 7:1:40:1.6, 5:1:30:1.2, 3:1:20:0.8, 1:1:10:0.4, 1:3:20:0.8, 1:5:30:1.2, 1:7:40:1.6, and 1:9:50:2.

[0025] The preparation method of the mitoxantrone-hypericin ion-paired engineered nanoassemblies is as follows:

[0026] a. Mitoxantrone, hypericin, sodium cholesterol sulfate, and DSPE-PEG 2K Dissolved in dimethyl sulfoxide (DMSO);

[0027] b. Under ultrasound probe, the solution obtained in step a is slowly added dropwise to water to form a uniform nanocolloid solution, namely hypericin-mitoxantrone ion-paired engineered nanoassemblies.

[0028] A third aspect of the present invention provides the application of the aforementioned nanoassemblies in the preparation of synergistic antitumor drugs.

[0029] The beneficial effects of this invention include:

[0030] (1) This invention is the first to combine type I and type II ICD inducers. In tumor cells, type I ICD inducers exert a chemotherapeutic effect, while type II ICD inducers generate reactive oxygen species under laser irradiation to exert a phototherapeutic effect. Both inducers provide tumor antigens while killing tumor cells. At the same time, type I and type II ICD inducers induce the release of DAMPs through different mechanisms, thereby enhancing the immune system's recognition of tumor antigens. The efficient synergy of chemotherapy, phototherapy, and immunotherapy achieves good anti-tumor effects, reduces the toxic side effects of chemotherapy, and utilizes the "distant effect" of anti-tumor immunity to improve the shortcomings of phototherapy in being ineffective against deep or metastatic tumors. Taking the type I ICD inducer mitoxantrone and the type II ICD inducer hypericin as examples, the combined use of these ingredients to prepare nanoassemblies has a synergistic effect on tumor treatment.

[0031] (2) This invention combines ion pairing with a self-assembly strategy to prepare hyperoside-mitoxantrone ion-paired engineered nanoassemblies with small particle size and uniform particle size distribution. The preparation method is simple and easy to implement. This provides more options for developing co-delivery nanomedicine systems for drugs with different physicochemical properties. Attached Figure Description

[0032] Figure 1 A is a photograph of hypericin and mitoxantrone after they were assembled by nanoprecipitation and then subjected to ultrafiltration and centrifugation. Figure 1 B is a photograph of hypericin, mitoxantrone, and sodium cholesterol sulfate after being prepared into an assembly via nanoprecipitation and then subjected to ultrafiltration and centrifugation; the nanoassemblies are retained by the ultrafiltration membrane, while the free drugs are separated to the bottom of the ultrafiltration tube by centrifugation.

[0033] Figure 2 The inhibition fraction-combination exponential curves (relative to single-drug solutions) of the hyperoside-mitoxantrone ion-paired engineered nanoassemblies of Example 2 of this invention on CT26 cells under different laser intensities; a. 7.2 J / cm 2 b.18J / cm 2 ;

[0034] Figure 3The inhibition fraction-combination exponential curves (relative to single-drug nanoassemblies) of the hyperoside-mitoxantrone ion-paired engineered nanoassemblies of Example 2 of this invention on CT26 cells under different laser intensities; a. 7.2 J / cm 2 b.18J / cm 2 ;

[0035] Figure 4 The image shows the immunofluorescence intensity of the hypericin-mitoxantrone ion-paired engineered nanoassembly of Example 2 of this invention, inducing calreticulin translocation (a) and high-mobility histone 1 release (b) at the cellular level. *: P < 0.05, **: P < 0.01, ***: P < 0.001, and ****: P < 0.0001 (all two-tailed t-tests).

[0036] Figure 5 Figure 1 shows the in vivo antitumor experiment results of the hypericin-mitoxantrone ion-paired engineered nanoassembly of Example 2 of the present invention; a. Mouse body weight change, b. Tumor volume change; where *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001 (all two-tailed t-tests);

[0037] Figure 6 The figures show the mean optical density and mean positive staining area in mouse tumor tissue after treatment with the hypericin-mitoxantrone ion-paired engineered nanoassemblies of Example 2 of this invention; a. mean optical density of CRT; b. mean positive staining area of ​​CD4, c. mean positive staining area of ​​CD8, d. mean positive staining area of ​​FOXP3; where *: P<0.05, **: P<0.01, ***: P<0.001, and ****: P<0.0001 (all two-tailed t-tests). Detailed Implementation

[0038] The present invention will be further described in detail below with reference to the embodiments, but the invention is not limited to the scope of the embodiments described herein.

[0039] This embodiment investigated the formulation characterization of hypericin-mitoxantrone ion-paired engineered nanoassemblies and the highly efficient synergistic therapeutic effect of combining the type I ICD inducer mitoxantrone and the type II ICD inducer hypericin with in vivo and in vitro chemotherapy-phototherapy-immunotherapy.

[0040] Example 1

[0041] Synergistic cytotoxic effects of mitoxantrone solution (a type I ICD inducer) and hypericin solution (a type II ICD inducer).

[0042] The MTT assay was used to investigate the synergistic cytotoxicity of mitoxantrone solution and hypericin solution against mouse colon cancer cells (CT26) induced by chemotherapy and phototherapy. First, cell suspension was added to 96-well plates at a concentration of 2000 cells / well and incubated at 37°C with 5% CO2 for 24 hours to allow cell adhesion. After cell adhesion, a series of concentrations of mitoxantrone solution (MTO Solution, M Sol), hypericin solution (HY Solution, H Sol), and a hypericin-mitoxantrone mixed solution (HY-MTO Solution, HM Sol) were added, with HY to MTO molar ratios of 1:9, 1:7, 1:5, 1:3, 1:1, 3:1, 5:1, 7:1, and 9:1, and the cells were incubated for 4 hours. Subsequently, the cells were irradiated with laser of different intensities (595 nm, 7.2 J / cm²). 2 and 18J / cm 2 Place the sample in an incubator and continue incubating for 44 hours. Add 20 μL of MTT solution (5 mg / mL) to each well and continue incubating for another 4 hours. Discard the liquid and add 200 μL of DMSO to each well to dissolve the blue-purple formazan. Measure the absorbance of the solution at 570 nm using a microplate reader.

[0043] The half-maximal inhibitory concentrations (IC50) of M Sol, H Sol, and HM Sol were calculated using GraphPad Prism 8. 50 The combination index (CI) was calculated using CompuSyn software (Chou and Talalay method). Different molar ratios of HY and MTO were subjected to laser irradiation at different intensities (7.2 J / cm²). 2 and 18J / cm 2 Table 1 shows the cytotoxicity synergistic index results for CT26 cells at the half-maximal inhibitory concentration (MCI). Mixed solutions with molar ratios of HY and MTO of 1:3, 1:5, 1:7, and 1:9 exhibited a cytotoxicity synergistic index less than 1, demonstrating a synergistic effect of chemotherapy and phototherapy. The mixed solution with a molar ratio of HY and MTO of 1:5 showed the highest synergistic cytotoxicity. Under different intensities of laser irradiation (7.2 J / cm²), the cytotoxicity synergistic effect was observed. 2 and 18J / cm 2 Under these conditions, the synergistic effect of the mixed solution cytotoxic agents remains consistent.

[0044] Table 1. Laser irradiation at different intensities (7.2 J / cm²) 2 and 18J / cm 2 The combination index of hyperoside-mitoxantrone mixed solution at half-maximal inhibitory concentration (MCI)

[0045]

[0046] Example 2

[0047] Preparation of hypericin-mitoxantrone ion-paired engineered nanoassemblies

[0048] Accurately weigh hypericin and mitoxantrone (molar ratio 1:5); dissolve hypericin, mitoxantrone, and sodium cholesterol sulfate (molar ratio 1:5:30) in an appropriate amount of DMSO as the organic phase. Use deionized water as the aqueous phase in a suitable container. Slowly add the organic phase dropwise to the aqueous phase under ultrasonic probe conditions, and continue ultrasonication for an appropriate time. Figure 1 As shown in Figure A, hypericin and mitoxantrone cannot form nanoassemblies. After centrifugation through an 8000–14000 Da ultrafiltration membrane, the drugs remain at the bottom of the ultrafiltration tube, indicating that the drugs are dissolved in the solution. However, with the aid of sodium cholesterol sulfate and mitoxantrone forming ion pairs, hypericin and mitoxantrone can form stable nanoassemblies. After ultrafiltration centrifugation, the nanoassemblies are retained by the ultrafiltration membrane, and the liquid at the bottom of the ultrafiltration tube becomes clear. Figure 1 B).

[0049] Accurately weigh hypericin, mitoxantrone, sodium cholesterol sulfate, and methoxy polyethylene glycol 2000-distearate phosphatidylethanolamine (DSPE-PEG). 2K (The molar ratio is 1:5:30:0.2), dissolved in an appropriate amount of DMSO as the organic phase. Deionized water is used as the aqueous phase and placed in a suitable container. Under ultrasonication, the organic phase is slowly added dropwise to the aqueous phase, and ultrasonication is continued for an appropriate time to form hypericin-mitoxantrone ion-paired engineered nanoassemblies (HY-MTO Nanoassemblies, HM NAs). Simultaneously, single-loaded hypericin nanoassemblies (HY Nanoassemblies, HNAs) and single-loaded mitoxantrone nanoassemblies (MTO Nanoassemblies, M NAs) are prepared by accurately weighing hypericin or mitoxantrone, sodium cholesterol sulfate, and methoxy polyethylene glycol 2000-distearate phosphatidylethanolamine (DSPE-PEG). 2K (The molar ratio was 1:5:0.2), dissolved in an appropriate amount of DMSO as the organic phase. Deionized water was used as the aqueous phase and placed in a suitable container. The organic phase was slowly added dropwise to the aqueous phase under ultrasonic probe, and ultrasonication was continued for an appropriate time. The particle size, particle size distribution, potential, and encapsulation efficiency of the ion-paired engineered nanoassemblies are shown in Table 2.

[0050] Table 2. Particle size, particle size distribution, potential, and encapsulation efficiency of ion-paired engineered nanoassemblies

[0051]

[0052] Encapsulation efficiency determination method: Nanoparticles and free drug were separated by ultrafiltration centrifugation, and then quantified by high performance liquid chromatography.

[0053] The results show that the particle size of HM NAs and M NAs is approximately 110 nm, while the particle size of H NAs is approximately 150 nm. The potentials of HM NAs, MNAs, and H NAs are around -40 mV. For MTO and HY, the encapsulation efficiency of all nanoassemblies exceeds 97%.

[0054] Example 3

[0055] Cytotoxic Synergistic Effects of Hypericin-Mitothrone Ion-Paired Engineered Nanoassemblies

[0056] The MTT assay was used to investigate the synergistic cytotoxicity of single-drug solutions and nanoassemblies (M Sol, H Sol, M NAs, H NAs) and dual-drug solutions and nanoassemblies (HM Sol, HM NAs) against mouse colon cancer cells (CT26). First, cell suspension was added to 96-well plates at a concentration of 2000 cells / well and incubated at 37°C, 5% CO2 for 24 hours to allow cell adhesion. After cell adhesion, a series of concentrations of M Sol, H Sol, M NAs, H NAs, H-MSol, and HM NAs (10, 50, 100, 200, 500, 1000, 2000, 4000 nmol / L) were added, with a HY to MTO molar ratio of 1:5, and the plates were incubated for 4 hours. Subsequently, the cells were irradiated with lasers of different intensities (595 nm, 7.2 J / cm²). 2 and 18J / cm 2 The cells were incubated for 44 hours. 20 μL of MTT solution (5 mg / mL) was added to each well, and incubation continued for another 4 hours. The liquid was discarded, and 200 μL of DMSO was added to each well to dissolve the blue-purple formazan. The absorbance of the solution was measured at 570 nm using a microplate reader. The combination index of HM Sol and HM NAs was calculated using CompuSyn software (Chou and Talalay method). Using the relative cell viability of the single-drug solution as a control, HM Sol and H-MNAs were subjected to laser irradiation at different intensities (7.2 J / cm²). 2 and 18J / cm 2 The inhibition fraction-combination exponential curve of CT26 cells is shown below. Figure 2 Using the relative cell viability of single-drug nanoassemblies as a control, HM NAs were subjected to laser irradiation at different intensities (7.2 J / cm²). 2 and 18J / cm 2 The inhibition fraction-combination exponential curve of CT26 cells is shown below. Figure 3 Both HM Sol and HM NAs exhibited a good synergistic effect on cytotoxicity, and these results validated our hypothesis that the combination of hypericin and mitoxantrone can effectively eliminate tumor cells through synergistic phototherapy and chemotherapy.

[0057] Example 4

[0058] Evaluation of the in vitro immunogenic cell death effect induced by hypericin-mitoxantrone ion-paired engineered nanoassemblies

[0059] Calreticulin (CRT) and high mobility group box 1 (HMGB 1) are considered major DAMP molecules responsible for immunogenic cell death. CRT acts as an "eat me" signal and disperses on the cell membrane after ICD. HMGB1 is initially localized to the nucleus and then translocates to the extracellular matrix during ICD. Their secretion and exposure involve gradual cell swelling, ultimately leading to cell membrane lysis, thereby releasing cellular contents and activating an immune response. CRT on the surface of CT26 cells was assessed by immunofluorescence. CT26 cells (5 × 10⁻⁶) were used. 4 Cells were seeded per well into 24-well plates covered with round coverslips. After culturing for 12 hours, the cells were treated with H₂Sol, M₂Sol, H₂NAs, M₂NAs, HM₂Sol, and HM₂NAs, with the same total dose (1 μmol / L) for each group. After culturing for 4 hours, the cells were then treated with a 595 nm laser (7.2 J / cm²). 2After 4 hours of incubation, cells were washed twice with cold PBS, fixed with 4% paraformaldehyde for 20 minutes, and blocked with 5% skim milk powder / triple-buffered saline and Tween 20 (TBST) solution for 30 minutes. Cells were then incubated with CRT primary antibody (1:500) at 4°C for 2 hours, followed by staining with FITC-labeled secondary antibody (1:500) at 25°C for 3 minutes. After Hoechst 33342 staining, CRT expression in CT26 cells was imaged using a laser scanning confocal microscope. Intracellular HMGB1 was also evaluated using immunofluorescence, with the difference being that cells were treated with each formulation for 4 hours, irradiated with laser, incubated for 20 hours, washed twice with cold PBS, fixed with 4% paraformaldehyde for 20 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and then blocked with 5% skim milk powder / TBST solution for 30 minutes. Cells were then incubated with HMGB1 primary antibody (1:500) at 4°C for 2 hours, followed by staining with FITC-labeled secondary antibody (1:500) at 25°C for 30 minutes. After staining with Hoechst 33342, intracellular HMGB1 fluorescence images in CT26 cells were observed using a laser scanning confocal microscope. Semi-quantitative analysis of the fluorescence intensity of CRT and HMGB1 in the laser scanning confocal microscope images was performed using ImageJ software. The results are shown below. Figure 4 The results showed that in all treatment groups, immunofluorescence of CRT translocation on the cell membrane was significantly increased, while green immunofluorescence of HMGB1 in the nuclear region was significantly reduced, indicating that the cells underwent immunogenic cell death. Notably, cells treated with the dual-drug combination formulation HM Sol and HM NAs exhibited significantly higher levels of CRT translocation and HMGB1 release compared to single-drug solutions and nanoassemblies. This suggests that the combination of hyperoside and mitoxantrone can significantly amplify the immunogenic cell death effect and could serve as an ideal synergistic strategy for promoting antigen-specific immune responses.

[0060] Example 5

[0061] In vivo antitumor effects of hypericin-mitoxantrone ion-paired engineered nanoassemblies

[0062] Mouse colon cancer cell suspension (CT26, 10) 6 (100 μL / cell) was injected subcutaneously on the dorsal side of male BALB / c mice (weighing 18-22 g). The inoculation continued until the tumor volume reached 100 mm². 3Around 1000 mice were randomly divided into 6 groups of 3 mice each: saline group, M Sol group, H Sol (+laser) group, HM Sol (+laser) group, HM NAs (+laser) group, and HM NAs (-laser) group. The nanoassemblies used for drug administration were prepared in Example 2. Hypericin and mitoxantrone were administered at doses of 0.42 μmol / kg and 2.08 μmol / kg, respectively. The drugs were administered via tail vein every other day for a total of 4 administrations. Six hours after each intravenous injection, the tumors in the laser-treated groups were irradiated (595 nm, 420 J / cm²). 2 Tumor volume and body weight changes in mice were monitored daily. Results are as follows: Figure 5 As shown, M Sol, H Sol (+laser), HM Sol (+laser), and HM NAs (-laser) all slowed tumor growth. In contrast, HM NAs (+laser) exhibited the strongest inhibitory effect on tumor growth. Furthermore, the body weight of mice in the HM NAs (+laser) group did not change significantly, while the body weight of mice in the HM Sol group decreased sharply, indicating that HM NAs are safer than HM Sol.

[0063] Example 6

[0064] Hypericin-mitoxantrone ion-paired engineered nanoassemblies activate anti-tumor immunity.

[0065] Immunohistochemical staining was performed on tumor tissues of mice after antitumor treatment to detect calreticulin and helper T lymphocytes (CD4+) in the tumor site. + T cells), cytotoxic T lymphocytes (CD8) + T cells and regulatory T cells (Tregs). Specifically, tumor tissue was fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and routinely dewaxed, rehydrated, and antigen-repaired to block endogenous peroxidase activity. Then, it was blocked with 3% bovine serum albumin at 25°C for 30 minutes, followed by overnight incubation with primary antibodies against CRT, CD4, CD8, and FOXP3 at 4°C, and then incubated with a secondary antibody conjugated to horseradish peroxidase. Finally, it was incubated with 3,3'-diaminobenzidine, and the nuclei were reverse-stained with hematoxylin and scanned using an inverted microscope. The mean optical density of CRT and the mean positive staining area of ​​CD4, CD8, and FOXP3 were semi-quantitatively analyzed using ImageJ software. Figure 6 ).

[0066] Treatment with M Sol, H Sol (+laser), and HM NAs (-laser) increased CRT expression levels in mouse tumor tissues, indicating that hypericin-based phototherapy and mitoxantrone-based chemotherapy induced ICD effects, respectively. The HM Sol (+laser) treatment group showed no significant difference in CRT expression compared to the other groups, possibly due to rapid clearance of the solution. The HM NAs (+laser) treatment group exhibited the highest CRT expression level, highlighting the effectiveness of hypericin-mitoxantrone ion-paired engineered nanoassemblies in synergistically enhancing ICD. CD4 + T cells play a crucial role in adaptive immune regulation, CD8 + T cells directly eliminate tumor cells by releasing cytokines. Compared with the saline group, mouse tumor tissues treated with M Sol, H Sol (+laser), and H-MSol (+laser) showed significantly higher CD4 counts. + T cells and CD8 + The infiltration of T cells was significantly increased, indicating that both hypericin-based phototherapy-induced ICD and mitoxantrone-based chemotherapy-induced ICD can activate anti-tumor immunity. Notably, CD4+ was significantly increased in tumor tissues of mice treated with HM NAs(+laser). + T cells and CD8 + T cell infiltration was significantly increased, while the number of Tregs was greatly reduced. In summary, the hyperoside-mitoxantrone ion-paired engineered nanoassemblies can effectively inhibit tumor development through chemotherapy-phototherapy combination, while synergistically activating anti-tumor immune responses. These findings validate the advantages of combining type I and type II ICD inducers.

Claims

1. A nano-assembly for synergistic anti-tumor effects through chemotherapy, phototherapy, and immunotherapy, characterized in that, The nanoassembly is composed of a type I ICD inducer, a type II ICD inducer, a hydrophobic auxiliary counterion, and an amphiphilic lipid ion pairing; the type I ICD inducer is mitoxantrone, the type II ICD inducer is hypericin, the hydrophobic auxiliary counterion is sodium cholesterol sulfate, and the amphiphilic lipid is methoxy polyethylene glycol 2000-distearate phosphatidylethanolamine (DSPE-PEG). 2K The molar ratio of the type I ICD inducer, type II ICD inducer, hydrophobic auxiliary counterion, and amphiphilic lipid is 1:5:30:0.

2.

2. The method for preparing the nano-assembly according to claim 1, characterized in that, Includes the following steps: a. Dissolve a type ICD inducer, a type II ICD inducer, a hydrophobic auxiliary counterion, and an amphiphilic lipid in dimethyl sulfoxide (DMSO), wherein the molar ratio of the type I ICD inducer, the type II ICD inducer, the hydrophobic auxiliary counterion, and the amphiphilic lipid is 1:5:30:0.2; b. Under ultrasonic probe, the solution obtained in step a is slowly added dropwise to water to form a uniform nanocolloid solution, thus obtaining the nanoassembly.

3. The preparation method according to claim 2, characterized in that, In step b above, the ultrasonic power is 100-400W and the ultrasonic time is 1-15min.

4. The preparation method according to claim 2, characterized in that, In step b, the volume of water used is 8-20 times the volume of dimethyl sulfoxide (DMSO) used in step a.

5. The preparation method according to claim 2, characterized in that, The nanoassemblies obtained in step b have a particle size of 80–200 nm, uniform particle size distribution, and a drug loading of 5–30%.

6. The preparation method according to claim 2, characterized in that, The nanoassembly is a hyperoside-mitoxantrone ion-paired engineered nanoassembly.

7. The use of the nanoassembly according to claim 1 in the preparation of a synergistic anti-colon cancer drug.

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