A drug delivery system for enhancing tumor immunotherapy and its preparation and application

Abstract

The present invention discloses a drug delivery system for enhancing tumor immunotherapy, as well as its preparation and application, and belongs to the field of biomedicine. The drug delivery system for enhancing tumor immunotherapy of the present invention is a platelet-exosome hybrid liposome (DCHL) that co-loads an AIE photosensitizer and chloroperoxidase (CPO). The drug delivery system for enhancing tumor immunotherapy of the present invention loads CPO and the novel AIE photosensitizer DPDPy and delivers them to the tumor site, enabling sustained singlet oxygen generation and enhanced EDT and tumor immunotherapy. The present invention provides a new solution for tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a drug delivery system for enhancing tumor immunotherapy, and its preparation and application. Background Art

[0002] Immunotherapy has become the fourth most important treatment for tumors after surgery, radiotherapy, and chemotherapy, and is one of the most advanced research areas in both applied research and clinical practice in recent years. Furthermore, with the rapid advancement of research related to the mechanisms of immunotherapy, it has been successfully applied to the clinical treatment of cancer. Reactive oxygen species-based anti-tumor therapies (such as photodynamic therapy, radiotherapy, chemodynamic therapy, and sonodynamic therapy) can effectively induce immunogenic cell death (ICD) of tumor cells and T cell infiltration, leading to a systemic immune response. This has been confirmed by numerous previous studies and constitutes a very effective approach to promote tumor immunotherapy efficacy. Among them, photosensitizers with aggregation-induced emission (AIEgens) can effectively generate reactive oxygen species (ROS) to kill tumor cells under light conditions and have been developed for cancer treatment. Furthermore, AIEgens have the advantages of simple preparation, resistance to photobleaching, excellent fluorescence properties, and good biocompatibility. They have been widely used in the field of photodynamic therapy (PDT), have made great progress in recent years, and have potential medical applications. However, ROS generally have a short lifespan (<0.04 μs) and are rapidly annihilated after the photoreaction, resulting in a relatively limited range of action. Continuous illumination is required to generate large amounts of ROS, but long-term illumination can cause skin damage and other adverse side effects. Therefore, new strategies that allow for the sustained generation of ROS at the tumor site are needed to promote the efficacy of PDT and immunotherapy.

[0003] Enzyme dynamic therapy (EDT) is a potential candidate therapy for promoting tumor immunotherapy. Chloroperoxidase (CPO) can catalyze the reaction of chloride with hydrogen peroxide (H2O2) to generate hypochlorous acid (HClO), forming singlet oxygen ( 1 The EDT process is limited by the content of hydrogen peroxide. Limited hydrogen peroxide content in cells and overexpression of glutathione will inhibit the anti-tumor effect of EDT. Therefore, it is necessary to change the tumor microenvironment to enhance the effect of EDT. Summary of the Invention

[0004] The purpose of the present invention is to address the deficiencies of the prior art and provide a drug delivery system for enhancing tumor immunotherapy and its preparation and application.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A drug delivery system for enhancing tumor immunotherapy is a platelet-exosome hybrid liposome co-loaded with an AIE photosensitizer and chloroperoxidase, named DCHL.

[0007] In some embodiments, the AIE photosensitizer is DPDPy having a structure shown in the following formula, and DPDPy has high singlet oxygen production capacity and photobleaching resistance.

[0008]

[0009] DPDPy is preferably prepared by a method comprising the following steps: dissolving 4-dimethylaminocinnamaldehyde and 1,4-lutidine iodide in a solvent, adding piperidine, and reacting under reflux to obtain DPDPy, wherein the solvent is preferably ethanol.

[0010] The preparation method of the drug delivery system for enhancing tumor immunotherapy is shown in the schematic diagram Figure 1 , including the following steps:

[0011] (1) 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), cholesterol (Cho), and AIE photosensitizer were dissolved in an organic solvent and formed into a thin film by rotary evaporation; a solution containing chloroperoxidase (CPO) was added to hydrate the film, and then ultrasonic treatment was performed to obtain liposomes co-loaded with AIE photosensitizer and chloroperoxidase.

[0012] (2) The liposomes co-loaded with AIE photosensitizer and chloroperoxidase were mixed with platelet exosomes, and platelet exosome hybrid liposomes (DCHL) co-loaded with AIE photosensitizer and chloroperoxidase were obtained by extrusion.

[0013] In the preparation method of the drug delivery system for enhancing tumor immunotherapy, the organic solvent is preferably chloroform; the rotary evaporation is preferably carried out at 30-70°C; the hydration is preferably carried out at 37°C; the solution containing chloroperoxidase is preferably a PBS solution containing chloroperoxidase, and the extrusion is preferably carried out using a polycarbonate membrane with a pore size of 100 nm.

[0014] Application of the drug delivery system for enhancing tumor immunotherapy in the preparation of anti-tumor drugs.

[0015] An anti-tumor drug comprises the drug delivery system for enhancing tumor immunotherapy, and may further comprise a pharmaceutically acceptable carrier or excipient.

[0016] The mechanism of action of the drug delivery system (DCHL) for enhancing tumor immunotherapy of the present invention is as follows Figure 1As shown, after DCHL targets tumor cells, the liposomes are destroyed by light, releasing DPDPy and CPO. DPDPy generates singlet oxygen, which leads to mitochondrial damage and the production of large amounts of hydrogen peroxide. Subsequently, intracellular chloride ions and hydrogen peroxide, under the action of CPO, can further generate hypochlorous acid and singlet oxygen, which continue to cause oxidative stress and immunogenic death of the tumor. Finally, tumor-associated antigens can stimulate the immune system, leading to dendritic cell (DC) maturation and T cell activation and infiltration, resulting in a systemic immune response.

[0017] The advantages and benefits of this invention include: Continuous generation of reactive oxygen species plays a key role in promoting tumor photoimmunotherapy. The drug delivery system for enhancing tumor immunotherapy of this invention loads and delivers CPO and the novel AIE photosensitizer DPDPy to the tumor site, enabling sustained singlet oxygen generation and enhancing EDT and tumor immunotherapy. This invention provides a new approach to tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the preparation of platelet-exosome hybrid liposomes co-loaded with AIE photosensitizer DPDPy and chloroperoxidase (CPO) and their application for uninterrupted singlet oxygen generation and enhanced tumor immunotherapy.

[0019] Figure 2 This is the performance evaluation of the AIE photosensitizer DPDPy. (A) PL spectra of DPDPy in toluene / water containing different concentrations of toluene. (B) Relationship between the αAIE value of DPDPy at 700nm and the different toluene concentrations in the toluene / water mixture. αAIE refers to the amount of toluene detected (f t ) with 0%f t (C) Decomposition rate of DPBF in the presence or absence of allergens and light irradiation, where A0 and A are the absorbance of DPBF at 420 nm before and after irradiation, respectively. (D) White light irradiation (0.1 W / cm 2 )Absorption spectra of DPDPy before and after 5 minutes.

[0020] Figure 3Characterization of platelet-exosome hybrid liposomes (DCHLs) co-loaded with DPDPy and CPO. (A) TEM images of DCL, PEV, and DCHL. (B) Diameter distribution of DCHL. (C) Hydrodynamic diameter and zeta potential of different formulations suspended in PBS. Data are presented as mean ± SD (n = 3). (D) Stability of DCHL in PBS or PBS containing 10% FBS. Data are presented as mean ± SD (n = 3). (E) PEV membranes labeled with fluorescent dyes (Dil and DiD) were fused with increasing amounts of liposomes (LIPs), and the fluorescence recovery rate of the donor (Dil) was measured to assess fusion. (F) Western blotting was used to detect PEV markers CD41 and P-selectin. (G) Absorption spectra of CPO (in PBS), DCHL (in PBS), blank PEV hybrid liposomes (BL, in PBS), and DPDPy (1% DMSO fraction). (H) Absorption spectra of DPBF treated with different formulations. (I) Absorption spectra of DPBF at different irradiation time points in the presence of DCHL. (J) Cumulative release curves of CPO under different conditions. Data are expressed as mean ± SD (n = 3). (K) CLSM images of cancer cells incubated with DiO-labeled DCL or DCHL for 1 h. Blue: DAPI; Red: DiO. Scale bar: 10 μm. (L) Figure 3 DiO fluorescence intensity in K cells, and PBS-treated cells served as controls.

[0021] Figure 4 DCHL can achieve uninterrupted singlet oxygen generation in tumor cells. (A) Fluorescence images of 4T1 cells treated with different formulations, stained with mitochondrial red fluorescent Violet and DAPI. Scale bar: 10 μm. (BC) Fluorescence imaging of H2O2 in 4T1 cells after different treatments and the corresponding fluorescence intensity (n=3). Scale bar: 10 μm. (DE) Fluorescence images of 4T1 cells treated with different formulations at 0 and 4 h after irradiation. 1 O2 fluorescence imaging and corresponding flow cytometry fluorescence intensity analysis. Scale bar: 10 μm. ***P < 0.005; Student's t-test.

[0022] Figure 5DCHL inhibits 4T1 cell growth and induces immunogenic cell death. (A) Cell viability of 4T1 cells after different treatments (n=3). (B) Cell viability of 4T1 cells after treatment with DCHL+L at different DPDPy concentrations (n=3). (C) GSH content in 4T1 cells after different treatments (n=3). (D) Quantitative analysis of HMGB1 release from 4T1 tumor cells 24 hours after various treatments. (E) Immunofluorescence staining (Green: CRT, Blue: DAPI) and fluorescence intensity of CRT-exposed 4T1 tumor cells after various treatments. Scale bar: 10 μm. **P<0.05, ***P<0.005; Student's t-test.

[0023] Figure 6 The circulation and tumor-targeting abilities of DCHL in tumor-bearing mice. (A) Pharmacokinetic profiles of DCL and DCHL in tumor-bearing mice. (B) Ex vivo images of tumor tissues and organs harvested from 4T1 tumor-bearing mice 24 hours after injection of DCL or DCHL.

[0024] Figure 7 The anti-tumor activity of DCHL in tumor-bearing mice. (A) Schematic diagram of 4T1 tumor treatment. (BC) Changes in primary and distant tumor volume in tumor-bearing mice after different treatments. (D) Tumor weights in mice treated with different treatments. (E) Distant tumor growth curve for each mouse. (F) Treatment-induced lymph node dendritic cell maturation and CD4 + / CD8 + Flow cytometric analysis of T lymphocytes. (G) Hematoxylin and eosinophilic syndrome (HE), sclerosing globulin (SOSG), and cytochrome P-terminal (CRT) staining of primary tumor tissues following different treatments. Scale bars: HE and SOSG, 40 μm; CRT, 20 μm. **P < 0.05, ***P < 0.005; Student's t-test.

[0025] Figure 8 The levels of proinflammatory cytokines IFN-γ (A) and TNF-α (B) in the serum of mice treated with different methods.

[0026] Figure 9 CD8 + The number of T cells.

[0027] Figure 10 DCHL has good biosafety. (A) Mouse body weight during administration. (B) Blood biochemical analysis of mice after DCHL treatment. (C) HE staining of major organs of mice after DCHL treatment.

[0028] Figure 11Figure 1: Effects of DCHL on 4T1 tumor rechallenge and recurrence. (A) Schematic diagram of 4T1 tumor rechallenge and recurrence. (B) Growth of recurrent 4T1 tumors. (C) Growth of rechallenge 4T1 tumors. (D) and (F) Flow cytometry analysis of central memory T cells (TCM, CD62L) in blood on day 14 after DCHL treatment. + CD44 + ) and CD3 + CD8 + Proportion of T cells. (E) Growth curve of reactivated tumors in each mouse. (G) TUNEL staining analysis of recurrent and reactivated tumor tissues after various treatments. Scale bar: 20 μm. Data are expressed as mean ± SD, **P < 0.01, ***P < 0.001; Student's t-test. DETAILED DESCRIPTION

[0029] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be considered equivalent replacement methods and shall be included in the scope of protection of the present invention.

[0030] Example 1 Preparation and Performance Evaluation of Platelet-Exosome Hybrid Liposomes (DCHL) Co-loaded with DPDPy and CPO I. Preparation of Platelet-Exosome Hybrid Liposomes (DCHL) Co-loaded with DPDPy and CPO

[0031] (1) Synthesis of DPDPy

[0032] The synthetic route of DPDPy is as follows:

[0033]

[0034] The specific steps were as follows: 4-Dimethylaminocinnamaldehyde (0.22 g, 1.28 mmol) and 1,4-lutidine iodide (0.27 g, 1.16 mmol) were dissolved in ethanol (10 mL). A few drops of piperidine were added, and the reaction mixture was refluxed for 5 h. After cooling to room temperature, the crude product was further purified by flash silica gel column chromatography eluting with dichloromethane and methanol (DCM:methanol = 20:1, v / v) to obtain DPDPy (0.16 g, 40% yield). 1H NMR (400MHz, CDCl3, δin ppm):8.77(d,J=6.6Hz,2H),7.75(d,J=6.5Hz,2H),7.50(m,1H),7.47(d,J=8.8Hz,2H),7 .02(d,J=15.4Hz,2H),6.83(m,3H),4.45(s,3H),3.05(s,6H).HRMS(MALDI-TOF(m / z):[M] + calcd for C 18 H 21 N2 + ,265.1699;found,265.1701.

[0035] DPDPy has AIE characteristics ( Figure 2 A and Figure 2 B) and good singlet oxygen generation ability ( Figure 2 C), and excellent photobleaching resistance ( Figure 2 D).

[0036] (2) Preparation of platelet exosomes (PEV)

[0037] Platelet exosomes were prepared according to the method described in the literature (Q.Ma, Q.Fan, J.Xu, J.Bai, X.Han, Z.Dong, X.Zhou, Z.Liu, Z.Gu, C.Wang, Calming Cytokine Storm in Pneumonia by Targeted Delivery of TPCA-1 Using Platelet-Derived Extracellular Vesicles, Matter 2020, 3, 287-301.). The preparation method includes the following steps: whole blood was collected from the sinuses of BALB / c mice, resuspended in PBS containing EDTA (5mM, Sigma-Aldrich) and PGE1 (1mM, MCE), and then centrifuged at 100g for 15min to remove red blood cells. The supernatant was collected and centrifuged at 8000g for 20min. The centrifuged precipitate was resuspended, activated with thrombin (2U / mL, Solarbio) for 30min, and centrifuged at 800g for 10min. The collected supernatant was further ultracentrifuged at 100,000 rpm for 2 h to obtain platelet exosomes (PEVs).

[0038] (3) Preparation of platelet-exosome hybrid liposomes (DCHL) co-loaded with DPDPy and CPO

[0039] 27 mg of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 2.8 mg of cholesterol (Chol) and 3 mg of DPDPy were dissolved in chloroform and then evaporated in a rotary evaporator at 55°C for 80 min to form a thin film. A PBS solution containing 4KU chloroperoxidase (CPO, purchased from Macklin reagent) was then added to hydrate the film at 37°C for 5 min, ultrasonicated, and then PEV (3 mg of protein) was added and repeatedly extruded through a polycarbonate membrane with a pore size of 100 nm. The resulting DCHL particles were dialyzed overnight in a dialysis bag (MWCO 300KD) to remove unencapsulated DPDPy and CPO. The CPO and DPDPy loadings were calculated by UV-visible spectroscopy using a UV-visible spectrophotometer Lambda35 (Perkin-Elmer). Loading = M drug / M DCHL , where M refers to mass.

[0040] Blank platelet-exosome hybrid liposomes (BL) were prepared in the same manner except that PBS was used instead of the drug.

[0041] Liposomes co-loaded with DPDPy and CPO (DCL) were prepared in the same manner except that PEV was removed.

[0042] DPDPy-loaded platelet-exosome hybrid liposomes (DHL) were prepared in the same manner except that CPO was removed.

[0043] 2. Characteristic Identification

[0044] The morphology of the prepared liposomes was examined by TEM. Figure 3 As shown in A, DCL exhibits a distinct liposome fingerprint structure and is relatively uniform in size; platelet-derived exosomes have a disc-shaped structure, and DCHL has a morphology similar to liposomes. The hydrated particle size and zeta potential of DCL, PEV, and DCHL were measured by DLS. Figure 3 As shown in B and 3C, the zeta potential of DCHL is about -15.3 mV, which is lower than that of DCL (about -8.2 mV), which may be due to the introduction of PEV membrane protein. DCHL has good stability in PBS or FBS ( Figure 3 D).

[0045] use Resonance energy transfer (FRET) was used to verify the fusion of synthetic liposomes (LIP) and PEV. The PEV membrane was labeled with a pair of FRET dyes, 1,1′-dioctadecyl-3,3,3′,3′tetramethylindocarbocyanine-4-chlorobenzenesulfonate (DiD) as the fluorescence donor and 1,1′-dioctadecyl-3,3,3′,3′tetramethylindocarbocyanine perchlorate (DiI) as the fluorescence acceptor, and then fused with LIP in increasing amounts. Figure 3 In E, as the amount of LIP increased, the fluorescence at 565 nm recovered, but gradually decreased at 670 nm, indicating that the lipids were inserted into the PEV membrane, resulting in a weakening of the FRET interaction. CD41 and P-selectin proteins were detected in both PEV and DCHL ( Figure 3 F), indicating that PEV and DCL were successfully fused. The UV-visible absorption spectrum of DCHL showed that the absorption peaks of CPO and DPDPy appeared in DCHL, indicating that DPDPy and CPO were successfully loaded ( Figure 3 G). BCA protein quantification and UV absorption detection showed that the drug loading efficiency (DLE) of DCHL for DPDPy and CPO was 82.3% and 34.7%, respectively.

[0046] The singlet oxygen generation of DCHL under different conditions was analyzed by DPBF degradation experiments. First, 0.05 mL of different formulas were added to 2 mL of DPBF: DCHL, PBS, DCHL with H2O2 and NaCl (DCHL+H2O2+NaCl), DCHL irradiated with white light (DCHL+L), and DCHL+L+H2O2+NaCl. The mixture was kept away from light for 30 minutes and then illuminated with white light (0.1 W / cm 2 ) as the light source, and measure the sample absorption spectrum at different times. Figure 3 H and Figure 3 As shown in Figure 1, DCHL significantly degraded DPBF under light. In the presence of hydrogen peroxide and sodium chloride, DCHL also significantly degraded DPBF, driven by CPO catalysis. Therefore, DCHL can efficiently generate singlet oxygen and is a potential nanomedicine for tumor therapy both in vitro and in vivo.

[0047] To study the stimulatory effect of laser irradiation on CPO release, 10 mL of DCHL containing 1 KU CPO was added to the culture dish with or without 0.1 W / cm 2 The CPO release experiment was conducted under white light irradiation (400-700nm) for 3min, and the released CPO was determined using the bicinchoninic acid (BCA) protein assay. Under light irradiation, CPO can be rapidly released ( Figure 3 This can be attributed to the disruption of the phospholipid bilayer caused by singlet oxygen generated by PDT.

[0048] 4T1 cells were seeded in 24-well plates and cultured for 12 h. Then, 100 μL of DiO-labeled DCL or DCHL (containing 0.01 mg DPDPy) was added to the culture medium. The cells were then incubated at 37°C, 5% CO2 for 1 h and washed three times with PBS. The cells were harvested, stained with DAPI, and imaged using a confocal laser scanning microscope (CLSM). Fluorescence intensity was measured by flow cytometry. Figure 3 As shown in K and 3L, after 1 h of incubation, the fluorescence of DPDPy in the tumor cells of the DCHL group was significantly stronger than that in the DCL group, indicating that DCHL can adhere to tumor cells in a very short time, which also provides a basis for the targeted treatment of DCHL under in vitro and in vivo conditions.

[0049] The above results indicate that the DCHL nanosystem can target tumor cells and has a good ability to produce singlet oxygen.

[0050] Example 2 In vitro anti-tumor ability of DCHL

[0051] 1. Experimental Methods

[0052] The cell line 4T1 mouse breast cancer cell line used in the experiment was cultured in RPMI-1640 medium containing 10% FBS in a humidified incubator at 37° C. and 5% CO 2 .

[0053] (1) Mitochondrial integrity assay

[0054] 4T1 cells were seeded in 6-well plates and cultured overnight. They were divided into groups and treated as follows and then cultured for 2 h: (1) PBS; (2) L (0.1 W / cm 2 , 5 min); (3) DPDPy + L; (4) DHL; (5) DHL + L. The DPDPy concentration was 10 μg / mL. After removing the residual nanomaterials, MitoTracker Red solution was added and incubated for 30 min. After removing the culture medium, the cells were washed three times with PBS, and fluorescence images were recorded using CLSM.

[0055] (2) Determination of intracellular H2O2

[0056] 4T1 cells were seeded in 6-well plates and cultured overnight. They were grouped and treated as follows and then cultured for 2 h: (1) PBS; (2) L (0.1 W / cm 2 , 5min); (3) DPDPy+L; (4) DHL; (5) DHL+L. DPDPy concentration was 10μg / mL. -6Cells were incubated with a H2O2 indicator (BES-H2O2-Ac) for 40 min. The culture medium was removed and the cells were washed three times with PBS. Fluorescence images were recorded using a CLSM.

[0057] (3) Intracellular ROS generation

[0058] 4T1 cells (1.5×10 5 / well) were inoculated in 12-well plates and cultured for 12 h. The groups were treated as follows: (1) PBS; (2) L (0.1 W / cm 2 , 5min); (3) DCHL; (4) DCL+L; (5) DHL+L; (6) DCHL+L. The DPDPy concentration was 10μg / mL. At 0 or 4h after irradiation, 10×10 -6 Cells were incubated with MSOSG (singlet oxygen sensor green fluorescent probe, purchased from Dalian Meilun Biotechnology Co., Ltd.) in RPMI-1640 for 10 min, and then the samples were observed under CLSM. Fluorescence intensity was detected by flow cytometry.

[0059] (4) DCHL induces tumor cell immunogenic death (ICD)

[0060] 4T1 cells were seeded in 48-well plates (2×10 4 After 12 hours, the cells were divided into groups and treated as follows: (1) PBS; (2) L (0.1W / cm 2 , 5min); (3) DCHL; (4) DCL+L; (5) DHL+L; (6) DCHL+L. The DPDPy concentration was 10μg / mL. The cells were cultured for another 24h, and 20μL of culture medium was used for HMGB1 (high mobility group protein B1) ELISA (HMGB1 ELISA kit purchased from Beijing Solarbio Technology Co., Ltd.) detection. The cells were then washed 3 times with PBS, fixed with 4% PFA, and permeabilized with 0.1% TritonX-100 for 10min. After washing 3 times with PBS, the cells were blocked with 10% FBS and incubated with anti-CRT antibody (Alexa Fluor 500). 647) and incubated for 30 min. The cells were washed three times with PBS and then stained with DAPI for 20 min. Finally, the cells were washed three times with PBS and observed using CLSM. Fluorescence intensity was measured using ImageJ software.

[0061] (5) In vitro anticancer effect of DCHL

[0062] 4T1 cells were incubated in a six-well plate at 37°C and 5% CO2 for 24 h. Afterwards, the culture medium was replaced with fresh culture medium and the cells were incubated in 6 different groups: (1) PBS; (2) L (0.1 W / cm 2 , 5 min); (3) DCHL; (4) DCL+L; (5) DHL+L; (6) DCHL+L. After incubation for another 48 h, cell viability was detected using an MTT cytotoxicity assay kit (Beyotime Biotech. Inc.).

[0063] 2. Results

[0064] After the successful preparation of DCHL, the ability of DCHL to damage mitochondria in tumor cells was studied, and MitoTrackerRed was used to investigate possible changes in mitochondrial membrane potential. Figure 4 As shown in A, the mitochondria in the PBS, L, and DHL groups showed significant red fluorescence, while the DHL+L and DPDPy+L groups showed almost no red fluorescence, indicating that the mitochondria in these two groups were damaged. Figure 4 B and Figure 4 As shown in C, the DHL+L and DPDPy+L groups produced significant hydrogen peroxide. Subsequently, CPO was introduced to study the continuous production of singlet oxygen by DCHL in tumors, as shown in Figure 4 D and Figure 4 As shown in E, DCL, DHL or DCHL can generate a large amount of singlet oxygen under light irradiation. However, after 4 hours, it was observed that only most cells in the DCHL group retained green fluorescence (SOSG). By flow cytometric analysis, after 4 hours, more than 70% of the cells in the DCHL+L group still maintained high levels of singlet oxygen, while a large number of cells in the DHL and DCL groups had low levels of singlet oxygen, and only a small number of cells still had high levels of singlet oxygen. For the DCL+L group, due to the lack of targeting ability of platelet exosomes, the concentration of CPO entering the tumor cells was low, and EDT could not be efficiently conducted to generate a large amount of singlet oxygen. For the DHL+L group, due to the lack of CPO-mediated EDT effect, it was impossible to maintain a high level of singlet oxygen for a long time. Therefore, DCHL can achieve uninterrupted singlet oxygen generation in tumor cells.

[0065] Furthermore, the growth inhibitory ability and immunogenic death-inducing effect of DCHL on 4T1 cells were studied. Figure 5 As shown in A, the tumor cell survival rate in the DCHL+L group was less than 10%, while that in the DHL+L group was close to 40%. This indicates that the introduction of CPO can significantly enhance the anti-tumor effect of DCHL. However, the tumor cells in the DCL+L group showed a higher survival rate, which may be due to weaker targeting ability and insufficient intracellular internalization. DCHL inhibited tumor cell growth in a concentration-dependent manner ( Figure 5B) Singlet oxygen can react with intracellular GSH to consume GSH, thereby promoting further oxidative damage to tumor cells. Figure 5 As shown in C, the intracellular GSH content in the DCHL+L group was the lowest, which may be due to the continuous production of singlet oxygen in the DCHL+L group. Intracellular GSH can participate in a variety of physiological activities and plays an important role in tumor recurrence and drug resistance. The better GSH consumption ability enables DCHL to promote the efficacy of radiotherapy, chemotherapy and other therapies. Next, the effect of DCHL on tumor immunogenic death was studied. Figure 5 As shown in Figures D-5E, high-mobility group protein B1 (HMGB1) is a highly conserved nuclear protein that can induce immunogenic cell death (ICD), enhance anti-tumor immunity, and exhibit significant anti-cancer effects. After DCHL+L treatment, tumor cells released more HMGB1 protein and exhibited significant CRT fluorescence, indicating that DCHL+L can induce immunogenic cell death in tumor cells, thus providing a solid foundation for subsequent T cell immunotherapy.

[0066] Example 3 In vivo anti-tumor ability of DCHL

[0067] 1. Experimental Methods

[0068] (1) Experimental animals were 5-6-week-old female BALB / c mice purchased from Weitonglihua Co., Ltd. (Beijing, China). Animal experiments were performed according to protocols approved by the Ministry of Health of the People's Republic of China and approved by the Animal Research Management Committee of Shenzhen People's Hospital.

[0069] (2) In vivo pharmacokinetics and distribution studies

[0070] 5×10 6 4T1 cells, when the tumor reaches 300mm 3 Balb / c mice (n=3) were intravenously injected with 100 μL of PBS containing DCL or DCHL (DPDPy equivalent dose of 10 mg / kg). At various time points after injection (i.e., 0.5, 1, 2, 4, 8, 24, and 48 hours), 20 μL of plasma was collected from the tail vein and centrifuged at 10,000 rpm for 10 minutes. The supernatant was collected and DPDPy concentration was quantified using a fluorescence spectrometer (FLS980).

[0071] 5×10 6 4T1 cells, when the tumor reaches 300mm 3Tumor-bearing mice (n=3) were intravenously injected with 100 μL of PBS containing DiR-labeled DCL or DCHL (DPDPy dose was 10 mg / kg). Mice were imaged using an IVIS system at 6, 12, and 24 hours after injection. Mice were then sacrificed 24 hours after injection, and tumors and major organs were harvested for imaging analysis and fluorescence intensity measurement using an IVIS system.

[0072] (3) Evaluation of oxidative stress in tumors

[0073] 5×10 6 4T1 cells, when the tumor reaches 300mm 3 The mice were randomly divided into 6 groups (3 mice in each group): (1) PBS; (2) L (0.1 W / cm 2 , 5 min); (3) DCHL; (4) DCL+L; (5) DHL+L; (6) DCHL+L. The DPDPy dose was 10 mg / kg. SOSG was intratumorally injected into the tumor tissue before light exposure. After laser irradiation, the tumor tissue was immediately removed for frozen section staining and observed using confocal laser scanning microscopy (CLSM).

[0074] (4) In vivo anti-tumor studies

[0075] 5×10 6 4T1 cells (primary tumor) were injected subcutaneously into the left flank. 6 4T1 cells (distant tumor). First, mice were randomly divided into 6 groups (5 mice in each group): (1) PBS; (2) L (0.1W / cm 2 , 5 min); (3) DCHL; (4) DCL+L; (5) DHL+L; (6) DCHL+L. The DPDPy dose was 5 mg / kg. The body weight and tumor volume of all groups of mice were monitored every 3 days. Tumor length and tumor width were measured using a caliper, and tumor volume was calculated according to the following formula: Tumor volume = tumor length × tumor width 2 / 2. After 15 days of treatment, the mice were killed. Blood samples were collected from these mice for blood biochemical analysis. The five major organs (heart, liver, spleen, lungs and kidneys) of all mice were harvested, washed with PBS, and fixed with paraformaldehyde for histological analysis. The tumor tissues were weighed, fixed with 4% neutral buffered formalin, processed with conventional paraffin, and sliced ​​at 4 μm. The primary lesion sections were then stained with HE and CRT, and finally examined using an optical microscope (BX51, Olympus, Japan) and a fluorescence microscope (IX81, Olympus, Japan).

[0076] (5) Study of in vivo immune response after different treatments

[0077] To detect DC maturation in vivo, inguinal lymph nodes (LN) were collected and immunofluorescently stained with anti-CD80-BV421 (BD Bioscience) and anti-CD86-APC (ab218757) antibodies. + The DC maturation rate in LN was examined using a cell sorting kit (Novo Biotechnology Co., Ltd.) and flow cytometry. To investigate the T cell content in distant tumors, tumors were harvested from mice in different groups and mouse CD3 T cells were used according to the manufacturer's protocol. + T cell sorting kit (Suzhou Beaver Biomedical Engineering Co., Ltd.), anti-CD8-Alexa Fluor 488 (ab237364) and anti-CD4-PE (ab252151) antibodies were used for processing. The distant tumor tissue was cut into small pieces and placed in a glass homogenizer containing PBS (pH 7.4) containing 2% heat-inactivated fetal bovine serum. Then, a single cell suspension was prepared by gently applying pressure with a homogenizer without adding digestive enzymes. Finally, CD3 + T cells were stained with fluorescently labeled antibodies after red blood cell removal using RBC lysis buffer. To analyze drug-induced cytokine secretion, whole blood was collected from mice 2 days after administration. Serum concentrations of proinflammatory cytokines (including TNF-α and IFN-γ) were then analyzed using ELISA kits (Neobioscience Co., Ltd., China) according to the manufacturer's instructions.

[0078] (6) Research on inhibiting tumor reactivation and recurrence

[0079] To investigate the anti-tumor reactivation and recurrence effects of DCHL, primary tumor-bearing mice were treated with the various treatments described above. After resection of the primary tumor, secondary 4T1 tumors were re-inoculated in the left hind leg of the mice. The growth of the secondary tumors was recorded at regular intervals. Blood samples were collected 14 days after the first administration for immune memory studies. + and CD8 + T cell isolation kit (NovoBiotechnology Co., LTD.) was used to isolate live CD3 + CD8 + T lymphocytes were stained with antibodies (anti-CD62L-BV421 and anti-CD44-APC-Cy7), and T cell subsets were analyzed by flow cytometry.

[0080] 2. Results

[0081] (1) In vivo anti-tumor activity of DCHL

[0082] Compared with DCL, DCHL has better long-term circulation ability and tumor targeting ability ( Figure 6 A and Figure 6 B), showing the advantages of fusogenic liposomes in drug delivery. Next, a bilateral breast tumor model was established by subcutaneously injecting 4T1 cells into the left and right regions of mice ( Figure 7 A). The left and right tumors are primary and distant tumors, respectively. Seven days after inoculation, mice were randomly divided into 6 groups (n = 5 per group): (1) PBS; (2) L (0.1 W / cm 2 , 5min); (3) injection of DCHL; (4) injection of DCL followed by light irradiation (DCL+L); (5) injection of DHL followed by light irradiation (DHL+L); (6) injection of DCHL followed by light irradiation (DCHL+L). Groups 4, 5, and 6 were exposed to white light only for 5min on the right tumor (primary lesion). During the treatment period, the tumor size was recorded every three days ( Figure 7 B and Figure 7 C), tumor weight Figure 7 D. After DCHL+L treatment, the growth of primary tumors was significantly inhibited, and the distant tumors in DCHL+L mice were also inhibited. The distant tumor growth curve of mice during treatment is shown in Figure 7 E.

[0083] The therapeutic mechanism of DCHL+L was further investigated by detecting dendritic cells (DC) in lymph nodes, T cell infiltration in distant tumors, and serum proinflammatory cytokines. DC plays a key role in immune response by participating in the initiation, regulation, and maintenance of innate and adaptive immune responses. T lymphocytes can bind to co-stimulatory molecules (CD80, CD86), which is an important indicator for inducing T cell-mediated immune responses and a marker of DC maturation. The maturation of lymph node dendritic cells and CD4 in distant tumors in different treatment groups were significantly different. + / CD8 + The results of flow cytometric analysis of T lymphocytes are shown in Figure 7 F. The percentage of mature DCs in the DCHL+L group was 50.3%, 1.47 times that of the DHL+L group (34.3%) and approximately 4.07 times that of the control group (9.93%). Serum cytokine concentrations were measured by enzyme-linked immunosorbent assay to evaluate the systemic immune response induced by DC maturation. Compared with the DCL+L and DHL+L groups, DCHL+L treatment consistently promoted the secretion of IFN-γ and TNF-α ( Figure 8 A and Figure 8 B) CD8 in distant tumors + The number of T cells increased significantly ( Figure 9), indicating that the combination of DCHL and laser therapy achieved a stronger anti-tumor immune response. In addition, SOSG was injected into the tumor 12 hours after laser irradiation of the primary lesion, and it was found that only the tumor sections of the DCHL+L group showed strong SOSG and CRT fluorescence ( Figure 7 G). HE results showed that the tumor tissue was significantly damaged. These may be the reasons why the DCHL+L group induced a better immune response. During the entire administration period, there was no significant weight loss in the mice ( Figure 10 A), there is no obvious damage to liver and kidney function and tissue ( Figure 10 B and Figure 10 C), indicating that DCHL has good biosafety.

[0084] (2) Analysis of in vivo immune memory effect

[0085] Immune memory is crucial for long-term tumor suppression and prevention of recurrence. Figure 11 As shown in A, tumor recurrence was observed and tumor rechallenge experiments were performed to reveal the anti-tumor immune memory effect induced by DCHL in primary 4T1 tumor-bearing mice. Figure 11 As shown in B, all mice experienced tumor recurrence. However, the tumor growth in the DCHL+L treated group was significantly slower than that in the other control groups. Next, the mice receiving different treatments were re-challenged with secondary tumors of the same type after surgical removal of the primary tumors. Figure 11 As shown in Figure C, compared with the PBS group, the benefits of DCHL, DCL+L, and DHL+L treatment in inhibiting secondary tumors were negligible. However, in mice receiving DCHL+L, secondary tumor growth was inhibited. These results indicate that DCHL+L treatment induced a long-lasting immune response against secondary tumors. Central memory T cells (TCM) survive for a long time in the body and can proliferate and differentiate into effective memory T cells after stimulation with tumor antigens, thus playing a key role in long-term anti-tumor responses. Therefore, the proportion of TCM was detected to study the immune memory effect. As Figure 11 D and Figure 11 As shown in Figure F, DCHL treatment resulted in a negligible increase in the proportion of TCMs compared to PBS treatment. In contrast, DCHL+L treatment resulted in a significant increase in the proportion of TCMs. Furthermore, in tumor sections from recurrent and reactivated tumors, TUNEL fluorescence was observed in both the DCHL+L and DHL+L groups, whereas tumors in the other groups showed little to no apoptosis. These results suggest that treatment with DCHL in combination with PDT and EDT elicits a robust immune memory response.

[0086] The results of the mouse subcutaneous bilateral tumor model, tumor recurrence and re-stimulation model experiments showed that DCHL treatment of the primary lesion can lead to substantial inhibition of distant tumors, and the number of activated DC cells in the mouse lymph nodes and CD8 + The DCHLs of this invention provide a new strategy for tumor treatment.

Claims

1. A platelet-exosome hybrid liposome co-loaded with an AIE photosensitizer and chloroperoxidase, characterized by: The AIE photosensitizer is DPDPy with the following structure: DPDPy is prepared by a method comprising the following steps: dissolving 4-dimethylaminocinnamaldehyde and 1,4-lutidine iodide in ethanol, adding piperidine, and performing a reflux reaction to obtain DPDPy; The platelet-exosome hybrid liposomes co-loaded with an AIE photosensitizer and chloroperoxidase are prepared by a method comprising the following steps: (1) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, cholesterol, and an AIE photosensitizer are dissolved in an organic solvent and formed into a thin film by rotary evaporation; a solution containing chloroperoxidase is added to hydrate the film, and then ultrasonic treatment is performed to obtain liposomes co-loaded with the AIE photosensitizer and chloroperoxidase; (2) The liposomes co-loaded with AIE photosensitizer and chloroperoxidase were mixed with platelet exosomes, and the platelet exosome hybrid liposomes co-loaded with AIE photosensitizer and chloroperoxidase were obtained by extrusion and dialysis, wherein the dialysis was performed in a dialysis bag with a molecular weight cut-off of 300 kD.

2. The method for preparing the platelet-exosome hybrid liposomes co-loaded with an AIE photosensitizer and chloroperoxidase according to claim 1, characterized in that: The steps include: (1) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, cholesterol, and an AIE photosensitizer are dissolved in an organic solvent and formed into a thin film by rotary evaporation; a solution containing chloroperoxidase is added to hydrate the film, and then ultrasonic treatment is performed to obtain liposomes co-loaded with the AIE photosensitizer and chloroperoxidase; (2) The liposomes co-loaded with AIE photosensitizer and chloroperoxidase were mixed with platelet exosomes, and the platelet exosome hybrid liposomes co-loaded with AIE photosensitizer and chloroperoxidase were obtained by extrusion and dialysis, wherein the dialysis was performed in a dialysis bag with a molecular weight cut-off of 300 kD.

3. The method for preparing platelet-exosome hybrid liposomes co-loaded with an AIE photosensitizer and chloroperoxidase according to claim 2, characterized in that: In step (1), the organic solvent is chloroform; the rotary evaporation is performed at 30-70° C.; the hydration is performed at 37° C.; and the solution containing chloroperoxidase is a PBS solution containing chloroperoxidase.

4. The method for preparing platelet-exosome hybrid liposomes co-loaded with an AIE photosensitizer and chloroperoxidase according to claim 2, characterized in that: In step (2), the extrusion is performed using a polycarbonate membrane with a pore size of 100 nm.

5. Use of the platelet-exosome hybrid liposome co-loaded with an AIE photosensitizer and chloroperoxidase according to claim 1 in the preparation of an anti-tumor drug, characterized in that: The tumor is breast cancer.

6. An anti-tumor drug, characterized in that: The platelet-exosome hybrid liposome comprises the AIE photosensitizer and chloroperoxidase-loaded platelet according to claim 1; the tumor is breast cancer.

7. The antitumor drug according to claim 6, characterized in that: Contains a pharmaceutically acceptable carrier.

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