I-type and II-type immunogenic cell death inducer ion pairing engineered nano assembly and construction and application of I-type and II-type immunogenic cell death inducer ion pairing engineered nano assembly

Abstract

The invention belongs to the field of new auxiliary materials and new dosage forms of pharmaceutical preparations, and particularly relates to an I-type and II-type immunogen cell death inducer ion pairing engineering nano assembly and construction and application thereof. The nano assembly is formed by pairing an I-type ICD inducer, an II-type ICD inducer, hydrophobic auxiliary counter ions and amphiphilic lipid ions; the I-type ICD inducer is selected from mitoxantrone, adriamycin, oxaliplatin or cyclophosphamide; the type II ICD inducer is selected from hypericin; the hydrophobic auxiliary counter ions are selected from sodium cholesterol sulfate, cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, deoxycholic acid lysine derivatives, oleanolic acid and ursolic acid. The I-type ICD inducer and the II-type ICD inducer are combined for use, and the synergistic tumor treatment effect is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of new excipients and new dosage forms of pharmaceutical preparations, and relates to the ion-pairing engineering nanoscale assembly of type I and type II immunogenic cell death inducers and its construction, specifically to the construction of an ion-pairing engineering nanoscale assembly of a chemotherapeutic drug inducing immunogenic cell death and a photosensitizer inducing immunogenic cell death, and the synergistic anti-tumor effect provided by the combined chemotherapy, phototherapy and immunotherapy. Background Art

[0002] Malignant tumors seriously threaten human health. Traditional treatment methods (surgery, chemotherapy and radiotherapy) are highly invasive, have severe toxic and side effects, and are prone to multi-drug resistance, and cannot effectively inhibit the recurrence and metastasis of tumors. In recent years, new anti-tumor strategies represented by immunotherapy have developed rapidly and shown bright clinical application prospects. However, immunotherapy is only effective for a small number of tumor patients with immunogenic characteristics (≈10 - 30%), so improving the immunogenicity of tumors is crucial for enhancing anti-tumor immunotherapy.

[0003] Immunogenicity depends on antigenicity and adjuvanticity. Inducing immunogenic cell death (ICD) in tumor cells through chemotherapy, radiotherapy, phototherapy, etc. can generate tumor antigens through cytotoxic effects, and at the same time release damage-associated molecular patterns (DAMPs) to play an adjuvant role, thereby stimulating the body to produce a specific immune response and inhibiting the growth, metastasis and recurrence of tumors. According to the different mechanisms of inducing DAMPs, ICD inducers can be divided into type I and type II. Type I ICD inducers directly act on cytoplasmic proteins or cell membrane channels, or indirectly act on the endoplasmic reticulum through a series of metabolic pathways. While type II ICD inducers directly act on the endoplasmic reticulum and cause endoplasmic reticulum stress response by changing endoplasmic reticulum homeostasis. Currently, the research mainly focuses on using a single type of ICD inducer, and the effect is often limited, and it is mostly used in combination with other immunotherapies. Combining type I and type II ICD inducers is expected to synergistically kill tumor cells to produce antigens, and promote the release of DAMPs through multiple mechanisms, thereby enhancing the immune system's recognition of tumor antigens. In addition, the combined application of type I and type II ICD inducers is expected to achieve efficient synergy of different treatment modes (chemotherapy - phototherapy - immunotherapy). However, there are obvious differences in the physicochemical properties between type I and type II ICD inducers, which poses a challenge to co-loading them into a single drug delivery system. Therefore, there is an urgent need to construct a drug delivery platform for co-loading type I and type II ICD inducers.

[0004] In recent years, the wide application of small molecule self-assembled nano drug delivery systems in the biomedical field has greatly enriched drug delivery and tumor treatment strategies. According to the structural characteristics of some drugs containing ionizable groups, the present invention proposes a new strategy that combines hydrophobic ion pairing with self-assembled nano drug delivery systems. This method uses hydrophobic counterions to form ion pairs with ionizable drugs through electrostatic interactions, and self-assembly is achieved by balancing the interactions between the two. This ion-paired engineered nanoassembly provides the possibility for the co-delivery of drugs with different physicochemical properties to achieve the efficient co-loading of type I and type II ICD inducers. Summary of the Invention

[0005] The present invention designs and constructs type I and type II ICD inducer ion-paired engineered nanoassemblies with a synergistic effect of inducing immunogenic cell death, especially hypericin-mitoxantrone ion-paired engineered nanoassemblies, to achieve the 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 in the present invention can exert an efficient synergistic anti-tumor effect through the combination of chemotherapy and phototherapy. At the same time, the chemotherapeutic drug and the phototherapeutic drug can synergistically induce immunogenic cell death through different mechanisms to enhance anti-tumor immunity. The present invention will provide a new, safe and effective drug delivery strategy for combination therapy.

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

[0007] In the first aspect of the present invention, there is provided a nanoassembly for synergistic anti-tumor of chemotherapy, phototherapy and immunotherapy, which 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 cholesterolsulfate (SCS), cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, glycocholate, taurochenodeoxycholate, deoxycholic acid lysine derivative, oleanolic acid, ursolic acid.

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

[0010] In the above technical scheme, further, 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, further, 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 of the present invention are nanoassemblies formed by type I and type II ICD inducers through non-covalent forces, such as electrostatic effects, hydrophobic effects, intermolecular hydrogen bonds, etc.;

[0013] Specifically, ionizable drugs are used to form ion pairing with auxiliary counterions. The ion pairing strategy shields the inherent charge of the drug molecules and expands the hydrophobic region of the drug molecules to improve the physicochemical properties of the drug molecules, and ultimately achieves self-assembly by utilizing the interaction balance between the drug molecules and the auxiliary molecules.

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

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

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

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

[0018] In the above technical solution, further, in the above 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 described in step a is 1:(0.1-10):(1-10):(0.1-1);

[0020] Furthermore, the molar ratio of type I ICD inducer: type II ICD inducer: hydrophobic co - 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 I ICD inducer: type II ICD inducer: hydrophobic co - 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, 1:9:50:2, better co - assembled nanoparticles can be formed.

[0022] In the above - mentioned technical solution, furthermore, the preferred type I and type II ICD inducer ion - paired engineered nano - assemblies of the present invention are hypericin - mitoxantrone ion - paired engineered nano - assemblies. The particle size of the hypericin - mitoxantrone ion - paired engineered nano - assemblies described in step b is 80 - 200 nm, and the particle size distribution is uniform. The drug - loading amounts of mitoxantrone and hypericin are 5 - 30% respectively.

[0023] The hypericin - mitoxantrone ion - paired engineered nano - assemblies are formed by forming an ion pair between mitoxantrone and the hydrophobic co - counterion sodium cholesteryl sulfate, shielding the inherent charge of mitoxantrone, expanding the hydrophobic region of mitoxantrone, and finally forming nano - assemblies by the balance of non - covalent forces such as electrostatic force, hydrophobic force, and intermolecular hydrogen bond between mitoxantrone, hypericin and the auxiliary molecule sodium cholesteryl sulfate.

[0024] Among them, the molar ratio of mitoxantrone: hypericin: sodium cholesteryl sulfate: DSPE - PEG 2K 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, 1:9:50:2.

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

[0026] a. Dissolve mitoxantrone, hypericin, sodium cholesteryl sulfate and DSPE - PEG 2K in dimethyl sulfoxide (DMSO);

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

[0028] The third aspect of the present invention provides the use of the aforementioned nano-assemblies in the preparation of a synergistic anti-tumor drug.

[0029] The beneficial effects of the present invention include:

[0030] (1) For the first time, the present invention combines type I and type II ICD inducers. In tumor cells, the type I ICD inducer plays a role in chemotherapy, and the type II ICD inducer generates reactive oxygen species under laser irradiation to play a phototherapy role. Both provide tumor antigens while killing tumor cells. At the same time, the type I ICD inducer and the type II ICD inducer jointly induce the release of DAMPs through different mechanisms, thereby enhancing the immune system's recognition of tumor antigens. The highly efficient synergy of the three treatment modes of chemotherapy-phototherapy-immunotherapy achieves good anti-tumor effects, reduces the toxic and side effects of chemotherapy, utilizes the "abscopal effect" of anti-tumor immunity, and improves the disadvantage that phototherapy is difficult to take effect on deep or metastatic tumors; taking mitoxantrone as the type I ICD inducer and hypericin as the type II ICD inducer as examples, the combined preparation of nano-assemblies has a synergistic anti-tumor effect.

[0031] (2) The present invention combines the ion-pairing and self-assembly strategies to prepare hypericin-mitoxantrone ion-paired engineered nano-assemblies with smaller particle size and uniform particle size distribution, and the preparation method is simple and easy to operate. It provides more options for the development of co-delivery nano-drug delivery systems for drugs with different physicochemical properties. Description of the Drawings

[0032] Figure 1 A is a photograph after hypericin and mitoxantrone were prepared into assemblies by nano-precipitation and then ultrafiltration centrifugation; Figure 1 B is a photograph after hypericin, mitoxantrone and sodium cholesteryl sulfate were prepared into assemblies by nano-precipitation and then ultrafiltration centrifugation; the nano-assemblies were retained by the ultrafiltration membrane, while the free drugs were centrifuged to the bottom of the ultrafiltration tube;

[0033] Figure 2 is the inhibition fraction-combination index curve of the hypericin-mitoxantrone ion-paired engineered nano-assemblies of Example 2 of the present invention against CT26 cells under different laser intensities (relative to the single-drug solution); a. 7.2 J / cm 2 , b. 18 J / cm 2 ; Figure 3Inhibitory fraction - combination index curve of hypericin - mitoxantrone ion - paired engineered nano - assemblies in Example 2 of the present invention against CT26 cells under irradiation with different laser intensities (relative to single - drug nano - assemblies); a. 7.2 J / cm 2 , b. 18 J / cm 2 ;

[0034] Figure 4 Immunofluorescence intensity maps of hypericin - mitoxantrone ion - paired engineered nano - assemblies in Example 2 of the present invention inducing calreticulin translocation (a) and high - mobility group protein 1 release (b) at the cellular level, where *: P < 0.05, **: P < 0.01, ***: P < 0.001, and ****: P < 0.0001 (all are two - sided t - tests);

[0035] Figure 5 Antitumor experimental results in vivo of hypericin - mitoxantrone ion - paired engineered nano - assemblies in Example 2 of the present invention; a. Mouse body weight change graph, b. Tumor volume change graph; where *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001 (all are two - sided t - tests);

[0036] Figure 6 Results graphs of the average optical density and average positive staining area in mouse tumor tissues after treatment with hypericin - mitoxantrone ion - paired engineered nano - assemblies in Example 2 of the present invention; a. Average optical density of CRT; b. Average positive staining area of CD4, c. Average positive staining area of CD8, d. Average positive staining area of FOXP3; where *: P < 0.05, **: P < 0.01, ***: P < 0.001, and ****: P < 0.0001 (all are two - sided t - tests). Detailed implementation manners

[0037] The present invention will be further described in detail below in conjunction with the embodiments, but the invention is not limited to the scope of the described embodiments.

[0038] This embodiment studied the formulation characterization of hypericin - mitoxantrone ion - paired engineered nano - assemblies and the highly synergistic therapeutic effects of the in vitro and in vivo chemotherapy - phototherapy - immunotherapy triple - treatment modes of the combination of mitoxantrone, a type - I ICD inducer, and hypericin, a type - II ICD inducer.

[0039] Example 1

[0040] Cytotoxic synergy of mitoxantrone solution, a type - I ICD inducer, and hypericin solution, a type - II ICD inducer

[0041] The chemotherapeutic-photodynamic synergistic cytotoxicity of mitoxantrone solution and hypericin solution against mouse colon cancer cells (CT26) was investigated using the MTT method. First, cell suspension was added to a 96-well plate at a concentration of 2000 cells / well and incubated at 37 °C in a 5% CO 2 incubator for 24 hours to allow cell attachment. After cell attachment, serial concentrations of mitoxantrone solution (MTO Solution, M Sol), hypericin solution (HY Solution, H Sol), and hypericin-mitoxantrone mixed solution (HY-MTO Solution, H-M Sol) with molar ratios of HY to MTO of 1:9, 1:7, 1:5, 1:3, 1:1, 3:1, 5:1, 7:1, and 9:1 were added and incubated in the incubator for 4 hours. Subsequently, cells were irradiated with lasers of different intensities (595 nm, 7.2 J / cm 2 and 18 J / cm 2 ), incubated in the incubator for an additional 44 hours, 20 μL of MTT solution (5 mg / mL) was added to each well, and then incubated for another 4 hours. The liquid was discarded, 200 μL of DMSO was added to each well to dissolve the purple formazan, and the absorbance value of the solution was measured at a wavelength of 570 nm using a microplate reader.

[0042] The half-maximal inhibitory concentration (IC 50 ) values of M Sol, H Sol, and H-M Sol were calculated using GraphPad Prism 8. The combination index (CI) was calculated using CompuSyn software (Chou and Talalay method). The combination index results of HY and MTO at different molar ratios under different intensities of laser irradiation (7.2 J / cm 2 and 18 J / cm 2 ) at the half-maximal inhibitory concentration for CT26 cells are shown in Table 1. The cytotoxic combination index of the mixed solutions with molar ratios of HY to MTO of 1:3, 1:5, 1:7, and 1:9 was less than 1, indicating a chemotherapeutic-photodynamic synergistic effect. The mixed solution with a molar ratio of HY to MTO of 1:5 showed the highest cytotoxic synergistic effect. The cytotoxic synergistic effect of the mixed solution remained consistent under different intensities of laser irradiation (7.2 J / cm 2 and 18 J / cm 2 ).

[0043] Table 1 Combination index of hypericin-mitoxantrone mixed solution at the half-maximal inhibitory concentration under different intensities of laser irradiation (7.2 J / cm 2 and 18 J / cm 2 )

[0044]

[0045] Example 2

[0046] Preparation of Hypericin-Mitoxantrone Ion-Pair Engineered Nanoassemblies

[0047] Weigh precisely hypericin and mitoxantrone (molar ratio 1:5); hypericin, mitoxantrone, and sodium cholesteryl sulfate (molar ratio 1:5:30) were dissolved in an appropriate amount of DMSO to serve as the organic phase. Deionized water was used as the aqueous phase and placed in a suitable container. Under probe sonication, the organic phase was slowly added dropwise to the aqueous phase and sonicated for an appropriate additional time. As shown in Figure 1 A, hypericin and mitoxantrone alone could not form nanoassemblies. After centrifugation through an 8000–14,000 Da ultrafiltration membrane, the drugs were at the bottom of the ultrafiltration tube, indicating that the drugs were dissolved in the solution. However, by forming an ion pair with sodium cholesteryl sulfate and mitoxantrone, hypericin and mitoxantrone could form stable nanoassemblies. After ultrafiltration centrifugation, the nanoassemblies were retained by the ultrafiltration membrane and the liquid at the bottom of the ultrafiltration tube was clear ( Figure 1 B).

[0048] Precisely weigh hypericin, mitoxantrone, sodium cholesteryl sulfate, and methoxypolyethylene glycol 2000-distearoyl phosphatidylethanolamine (DSPE-PEG 2K )(molar ratio 1:5:30:0.2), dissolve in an appropriate amount of DMSO to serve as the organic phase. Deionized water was used as the aqueous phase and placed in a suitable container. Under probe sonication, the organic phase was slowly added dropwise to the aqueous phase and sonicated for an appropriate additional time to form hypericin-mitoxantrone ion-pair engineered nanoassemblies (HY-MTO Nanoassemblies, H-M NAs). At the same time, prepare hypericin-loaded nanoassemblies (HY Nanoassemblies, HNAs) and mitoxantrone-loaded nanoassemblies (MTO Nanoassemblies, M NAs), that is, precisely weigh hypericin or mitoxantrone, sodium cholesteryl sulfate, and methoxypolyethylene glycol 2000-distearoyl phosphatidylethanolamine (DSPE-PEG 2K )(molar ratio 1:5:0.2), dissolve in an appropriate amount of DMSO to serve as the organic phase. Deionized water was used as the aqueous phase and placed in a suitable container. Under probe sonication, the organic phase was slowly added dropwise to the aqueous phase and sonicated for an appropriate additional time. The particle size, particle size distribution, zeta potential, and encapsulation efficiency of the ion-pair engineered nanoassemblies are shown in Table 2.

[0049] Table 2 Particle Size, Particle Size Distribution, Zeta Potential, and Encapsulation Efficiency of Ion-Pair Engineered Nanoassemblies

[0050]

[0051] Encapsulation efficiency determination method: The nanoparticles and free drugs were separated by ultrafiltration centrifugation, and then quantified by high performance liquid chromatography.

[0052] The results showed that: The particle sizes of H-M NAs and M NAs were about 110 nm, while that of H NAs was about 150 nm. The zeta potentials of H-M NAs, M NAs and H NAs were around -40 mV. For MTO and HY, the encapsulation efficiency of all nanoassemblies exceeded 97%.

[0053] Example 3

[0054] Cytotoxic synergy of hypericin-mitoxantrone ion-paired engineered nanoassemblies

[0055] The chemo-photodynamic therapy synergistic cytotoxicity of single-drug solutions and nanoassemblies (M Sol, H Sol, M NAs, H NAs) as well as dual-drug combination solutions and nanoassemblies (H-M Sol, H-M NAs) against mouse colon cancer cells (CT26) was investigated by the MTT method. First, the cell suspension was added to a 96-well plate at a concentration of 2000 cells / well, and incubated at 37 °C in a 5% CO 2 incubator for 24 hours to allow cell attachment. After the cells attached, serial concentrations of M Sol, H Sol, M NAs, H NAs, H-M Sol and H-M NAs (10, 50, 100, 200, 500, 1000, 2000, 4000 nmol / L) were added, and the molar ratio of HY to MTO was 1:5, and incubated in the incubator for 4 hours. Subsequently, the cells were irradiated with lasers of different intensities (595 nm, 7.2 J / cm 2 and 18 J / cm 2 ), and incubated in the incubator for another 44 hours. Then, 20 μL of MTT solution (5 mg / mL) was added to each well, and incubated for another 4 hours. The liquid was discarded, 200 μL of DMSO was added to each well to dissolve the purple formazan, and the absorbance value of the solution was measured at a wavelength of 570 nm with an enzyme-linked immunosorbent assay reader. The combination index of H-M Sol and H-M NAs was calculated using CompuSyn software (Chou and Talalay method). Taking the relative cell viability of the single-drug solution as the control, the inhibition fraction-combination index curves of H-M Sol and H-M NAs against CT26 cells under different intensities of laser irradiation (7.2 J / cm 2 and 18 J / cm 2 ) are shown in Figure 2 . Taking the relative cell viability of the single-drug nanoassembly as the control, the inhibition fraction-combination index curves of H-M NAs against CT26 cells under different intensities of laser irradiation (7.2 J / cm 2 and 18 J / cm 2)Under the following conditions, the inhibition fraction - combination index curve of CT26 cells is shown in Figure 3 . Both H-M Sol and H-M NAs showed good cytotoxic synergy effects, and these results verified our hypothesis that the combination of hypericin and mitoxantrone could effectively eliminate tumor cells through synergistic phototherapy and chemotherapy.

[0056] Example 4

[0057] Evaluation of the effect of hypericin - mitoxantrone ion - paired engineered nanoassemblies on inducing immunogenic cell death in vitro

[0058] Calreticulin (CRT) and high mobility group box 1 (HMGB 1) are considered the main damage - associated molecular patterns (DAMPs) molecules that cause immunogenic cell death. CRT acts as an "eat me" signal and is dispersed on the cell membrane after the ICD process. HMGB1 is initially localized in the nucleus and then translocates to the extracellular matrix during the ICD process. Their secretion and exposure processes involve gradual cell swelling, eventually leading to cell membrane lysis, thereby releasing cell contents and activating the immune response. The CRT on the surface of CT26 cells was evaluated by immunofluorescence. CT26 cells (5×10 4 cells / well) were seeded into 24 - well plates coated with round coverslips. After culturing for 12 hours, the cells were treated with H Sol, M Sol, H NAs, M NAs, H-M Sol, and H-M NAs. The total dose of each group was the same (1 μmol / L). After culturing for 4 hours, a 595 nm laser (7.2 J / cm 2)Irradiation. After incubation for 4 hours, the cells were rinsed twice with cold PBS, fixed with 4% paraformaldehyde for 20 minutes, and blocked with 5% non-fat milk / TBST solution for 30 minutes. Then the cells were incubated with anti-CRT primary antibody (1:500) at 4 °C for 2 hours, and then stained with FITC-labeled secondary antibody (1:500) at 25 °C for 3 minutes. After Hoechst 33342 staining, the expression of CRT in CT26 cells was imaged by laser scanning confocal microscopy. The intracellular HMGB1 was also evaluated by immunofluorescence. The difference was that after the cells were treated with each group of preparations for 4 hours and irradiated with laser, they were incubated for 20 hours, and then the cells were rinsed 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% non-fat milk / TBST solution for 30 minutes. Subsequently, the cells were incubated with anti-HMGB1 primary antibody (1:500) at 4 °C for 2 hours, and then stained with FITC-labeled secondary antibody (1:500) at 25 °C for 30 minutes. After Hoechst 33342 staining, the fluorescence images of intracellular HMGB1 in CT26 cells were observed by laser scanning confocal microscopy. The fluorescence intensities of CRT and HMGB1 in the pictures taken by laser scanning confocal microscopy were semi-quantitatively analyzed using Image J software, and the results are shown in Figure 4 . The results showed that in all drug-administered groups, the immunofluorescence of CRT translocation on the cell membrane increased significantly, and the green immunofluorescence of HMGB1 in the nuclear region decreased significantly, indicating that the cells had undergone immunogenic cell death. Notably, the levels of CRT translocation and HMGB1 release in the cells treated with the dual-drug combination preparations H-M Sol and H-M NAs were significantly better than those of the single-drug solution and nano-assembly, which means that the combination of hypericin and mitoxantrone can significantly amplify the immunogenic cell death effect and can be used as an ideal synergistic strategy to promote antigen-specific immune responses.

[0059] Example 5

[0060] In vivo anti-tumor effect of hypericin-mitoxantrone ion-paired engineered nano-assemblies

[0061] A suspension of mouse colon cancer cells (CT26, 10 6 cells / 100 μL) was inoculated subcutaneously on the dorsal side of male BALB / c mice (body weight 18 - 22 g). When the tumor volume reached 100 mm 3Around, the tumor-bearing mice were randomly divided into 6 groups with 3 mice in each group, namely the normal saline group, the M Sol group, the H Sol(+laser) group, the H-M Sol(+laser) group, the H-M NAs(+laser) group, and the H-M NAs(-laser) group. The nanoassemblies used for administration were prepared in Example 2. The administration doses of hypericin and mitoxantrone were 0.42 μmol / kg and 2.08 μmol / kg, respectively. The drugs were administered via the tail vein once every 1 day for a total of 4 times. Six hours after each intravenous injection, the tumors in the laser treatment groups were irradiated (595 nm, 420 J / cm 2 ). The tumor volume and body weight changes of the mice were detected every day. The results are as Figure 5 shown. M Sol, H Sol(+laser), H-M Sol(+laser), and H-M NAs(-laser) could delay tumor growth. In contrast, H-M NAs(+laser) had the strongest inhibitory effect on tumor growth. In addition, there was no significant change in the body weight of the mice in the H-M NAs(+laser) group, while the body weight of the mice in the H-M Sol group decreased sharply, indicating that the safety of H-M NAs was better than that of H-M Sol.

[0062] Example 6

[0063] Effect of hypericin-mitoxantrone ion-paired engineered nanoassemblies on activating anti-tumor immunity

[0064] The tumor tissues of the mice after anti-tumor treatment were subjected to immunohistochemical staining to detect calreticulin, helper T lymphocytes (CD4 + T cells), cytotoxic T lymphocytes (CD8 + T cells), and regulatory T cells (Tregs) at the tumor sites. Specifically, the tumor tissues were fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and the sections were routinely dewaxed, rehydrated, antigen repaired, and endogenous peroxidase activity blocked. Then, they were blocked with 3% bovine serum albumin at 25 °C for 30 minutes, and subsequently incubated overnight with the primary antibodies against CRT, CD4, CD8, and FOXP3 at 4 °C, and then incubated with the secondary antibody conjugated with horseradish peroxidase. Finally, they were incubated with 3,3'-diaminobenzidine, counterstained with hematoxylin for the cell nuclei, and scanned using an inverted microscope. The average optical density of CRT and the average positive staining areas of CD4, CD8, and FOXP3 were semi-quantitatively analyzed using Image J software ( Figure 6 ).

[0065] After treatment with M Sol, H Sol(+laser) and H-M NAs(-laser), the expression level of CRT in the tumor tissues of mice increased, indicating that hypericin-based phototherapy and mitoxantrone-based chemotherapy induced ICD effects respectively. There was no significant difference in the CRT expression between the H-M Sol(+laser) treatment group and the above-mentioned groups, which might be due to the rapid clearance of the solution. The expression level of CRT in the H-M NAs(+laser) treatment group was the highest, highlighting the effectiveness of hypericin-mitoxantrone ion-pair engineered nanoassemblies in synergistically enhancing ICD. CD4 + T cells play a key role in adaptive immune regulation, and CD8 + T cells directly eliminate tumor cells by releasing cytokines. Compared with the saline group, the infiltration of CD4 + T cells and CD8 + T cells in the tumor tissues of mice treated with M Sol, H Sol(+laser) and H-MSol(+laser) increased significantly, indicating that ICD induced by hypericin-based phototherapy or mitoxantrone-based chemotherapy could activate anti-tumor immunity. Notably, the infiltration of CD4 + T cells and CD8 + T cells in the tumor tissues of mice in the H-M NAs(+laser) treatment group increased significantly, and at the same time, the number of Tregs also decreased greatly. In summary, hypericin-mitoxantrone ion-pair engineered nanoassemblies can effectively inhibit tumor development through the combination of chemotherapy and phototherapy, and simultaneously synergistically activate anti-tumor immune responses. These findings verify the advantages of the combination of type I and type II ICD inducers.

Claims

1. A nanoassembly for synergistic anti-tumor effects of 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 counter ion and an amphiphilic lipid ion pair; 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, cholic acid, deoxycholic acid, chenodeoxycholic acid, lithocholic acid, glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, deoxycholic acid lysine derivatives, oleanolic acid, and ursolic acid.

2. The nanoassembly for synergistic anti-tumor effects of chemotherapy, phototherapy and immunotherapy according to claim 1, characterized in that: The amphiphilic lipid is selected from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, methoxy polyethylene glycol 2000-distearate phosphatidylethanolamine (DSPE-PEG 2K ) or cholesterol-polyethylene glycol 2000 (CH-PEG).

3. The nanoassembly according to claim 1, characterized in that: 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).

4. The nanoassembly according to claim 1, characterized in that: The type I ICD inducer is selected from mitoxantrone, and the type II ICD inducer is selected from hypericin.

5. The method for preparing a nanoassembly according to any one of claims 1 to 4, characterized in that: The following steps are involved: a. dissolving a type I ICD inducer, a type II ICD inducer, a hydrophobic auxiliary counterion and an amphiphilic lipid in dimethyl sulfoxide (DMSO); b. Under probe ultrasound, slowly add the solution obtained in step a to water to form a uniform nanocolloid solution, thereby obtaining a nanoassembly.

6. The preparation method according to claim 5, characterized in that: In the above step b, the ultrasonic power is 100-400W, and the ultrasonic time is 1-15min.

7. The preparation method according to claim 5, characterized in that: In the step b, the volume of water used is 8-20 times the volume of dimethyl sulfoxide (DMSO) in step a.

8. The preparation method according to claim 5, characterized in that: The nano-assembly obtained in step b has a particle size of 80 to 200 nm, a uniform particle size distribution, and a drug loading of 5 to 30%.

9. The preparation method according to claim 5, characterized in that: The nanoassembly is a hypericin-mitoxantrone ion pairing engineered nanoassembly.

10. Use of the nanoassembly according to any one of claims 1 to 4 in the preparation of synergistic anti-tumor drugs.

Images