Composition for blocking excretion of lactic acid and enhancing tumor photoimmunotherapy as well as preparation method and application of composition

Abstract

The invention relates to a composition for blocking lactic acid efflux and enhancing tumor photoimmunotherapy as well as a preparation method and application of the composition, and the composition co-delivers an MCT4 inhibitor cyclostin and a photodiagnosis and treatment agent L8BO through a lactic acid metabolism checkpoint blocking strategy of a targeted monocarboxylic acid transporter 4 (MCT4) so as to enhance photoimmunotherapy. The composition L8-atSY has a strong intramolecular charge transfer effect, an optimized singlet-triplet energy gap and an excellent light capture capability, realizes simultaneous generation of I-type and II-type active oxygen, has a total active oxygen yield up to 503 times within the illumination time of 5 minutes, and also has photothermal conversion efficiency up to 46.2%. Under the trigger of lactic acid efflux blocking induction intracellular acidification, the nanoparticles based on the composition show an excellent tumor inhibition effect, almost completely inhibit primary tumors (the inhibition rate is 99.6%), significantly inhibit distal tumors (the inhibition rate is 68.6%), and reduce pulmonary metastasis at the same time.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a composition for blocking lactic acid efflux to enhance tumor photoimmunotherapy and a preparation method and application thereof. BACKGROUND

[0002] Phototherapy, including photodynamic therapy (PDT) and photothermal therapy (PTT), has attracted extensive attention due to its unique advantages, including non-invasiveness, low toxicity, precise spatiotemporal control, and less likelihood of developing therapeutic resistance. However, studies have shown that ROS-based PDT often has poor therapeutic effect, which is mainly because ROS can activate the protective autophagy of tumor cells. This adaptive response reduces the sensitivity of cells to ROS-induced damage, leads to immunosuppressive tolerance, and triggers subsequent drug resistance against ROS-dependent anti-tumor strategies. In addition, the inherent hypoxic microenvironment of solid tumors also seriously impairs the efficacy of PDT. Therefore, compared with singlet oxygen that depends on oxygen, designing photosensitizers that can efficiently generate reactive free radicals is crucial to improve the therapeutic effect of PDT. On the other hand, although the high temperature induced by PTT can destroy tumor cells, it is accompanied by the activation of heat shock proteins (HSPs), especially HSP70 and HSP90, to enhance the heat tolerance of tumor cells, thereby weakening the therapeutic effect. In addition, although elevated temperature can improve treatment efficiency, excessive heating has the risk of triggering inflammatory reactions and causing collateral damage to surrounding healthy tissues. These limitations highlight the urgent need for a synergistic PDT-PTT combined strategy to overcome the inherent shortcomings of single-mode treatment methods. However, the development of photosensitizers with both efficient free radical generation ability and high PCE still faces challenges. This is mainly due to the lack of suitable molecular structures and construction strategies, as well as the fierce competition between the energy dissipation pathways of PDT and PTT.

[0003] Efficient PDT and PTT can induce apoptosis of primary tumor cells and trigger immunogenic cell death (ICD) by releasing damage-associated molecular patterns (DAMPs), thus stimulating anti-tumor immune responses. Recent studies have found that photoimmunotherapy can effectively eradicate tumors and establish a long-lasting immune memory against recurrence. However, the therapeutic effect of photoimmunotherapy is still severely limited by insufficient infiltration of cytotoxic T lymphocytes and immunosuppressive tumor microenvironment (ITM). Among them, lactic acid is a key contributing factor to the characteristics of the ITM of solid tumors. The rapid growth, migration, invasion, immune escape and distant metastasis of tumor cells are based on metabolic reprogramming, which is characterized by energy production through glycolysis and accompanied by high lactic acid output, i.e. the "Warburg effect". This acidified microenvironment promotes the sustained survival of cancer cells and creates an ITM conducive to immune escape. Specifically, high concentrations of lactic acid directly inhibit the activity of natural killer (NK) cells and CD8 + T cells and induce their apoptosis. In contrast, regulatory T cells (Tregs) with immunosuppressive effects exhibit high lactic acid tolerance, allowing them to use lactic acid as an energy source and produce metabolic intermediates needed for proliferation. In addition, lactic acid drives macrophages to polarize to the pro-tumor M2 phenotype, thereby promoting tumor proliferation, migration and angiogenesis. Researchers have reported various strategies to regulate metabolic reprogramming of lactic acid in tumor cells, including inhibition of lactate dehydrogenase (LDH), monocarboxylate transporters (MCTs) and lactic acid receptor GPR81. Intratumoral infusion of sodium bicarbonate solution to neutralize and remove intratumoral lactic acid has also been explored. However, this method has limited effectiveness because systemically administered sodium bicarbonate lacks tumor specificity and requires continuous supplementation, hindering its clinical translation. Therefore, how to remodel the ITM by regulating lactic acid metabolism and enhance the efficacy of photoimmunotherapy to effectively inhibit tumor growth and metastasis is a key challenge that needs to be addressed. SUMMARY

[0004] The purpose of the present application is to provide a novel composition for enhancing photoimmunotherapy by targeting lactic acid metabolism-immune circuit reprogramming strategies, solving the problems existing in the prior art.

[0005] A composition for blocking lactic acid efflux to enhance tumor photoimmunotherapy, comprising Sylosin (SY) and a photo-diagnosis and therapy agent L8BO (abbreviated as L8).

[0006] L8BO is {2,2'-[(12,13-bis(2-butyloctyl)-12,13-dihydro-[1,2,5]thiadiazepino[3,4-e]thieno[2'',3':4',5']thieno[2',3':4,5]pyrrolo[3,2-g]thieno[2',3':4,5]thieno[3,2-b]indol-2,10-diyl)bis(methylylidene)]bis(5,6-dichloro-3-oxo-2,3-dihydro-1H-inden-2,1- ylidene)}dimalononitrile.

[0007] Syrosingopine (SY) is used to inhibit the activity of lactate transporter MCT4, and after the inactivation of MCT4, the efflux of lactic acid is prevented, thereby causing cell acidosis.

[0008] The L8BO in the composition of the present application is mainly responsible for the synergistic treatment of photodynamic and photothermal, but this treatment is limited by the acidic tumor inhibitory microenvironment, so syrosingopine SY is introduced. SY can reduce the expression of MCT4 lactate transporter, thereby inhibiting the efflux of lactic acid, leading to cell acidosis, thereby enhancing PDT and PTT.

[0009] Further, the mass ratio of the syrosingopine and the photodiagnosis and therapy agent L8BO is 1:(1-5). More preferably, the mass ratio of the syrosingopine and the photodiagnosis and therapy agent L8BO is 1:2.5.

[0010] The preparation method of the nanoparticles of the composition for inhibiting the efflux of lactic acid to enhance tumor photodynamic therapy, comprises the following steps:

[0011] (1) adding syrosingopine and photodiagnosis and therapy agent L8BO and DSPE-SS-PEG2000 into tetrahydrofuran solution;

[0012] (2) adding deionized water, and then ultrasonic treatment for 1-30 minutes.

[0013] DSPE-SS-PEG is a carrier for loading L8BO and syrosingopine, and the -SS- (disulfide bond) here can respond to the high expression of glutathione in tumor cells to release syrosingopine. It is found in the exploration of the present application that other carriers cannot be released responsively.

[0014] Further, the volume ratio of the added tetrahydrofuran solution and deionized water is 1:9.

[0015] The application of the composition for blocking the efflux of lactic acid to enhance tumor photodynamic therapy is used for preparing a photodynamic-photothermal therapy preparation for blocking the efflux of lactic acid.

[0016] The composition of the present application can achieve a lactic acid metabolism checkpoint blocking strategy targeting monocarboxylate transporter 4 (MCT4), co-deliver MCT4 inhibitor xilonixapam (SY) and photodiagnostic agent L8BO (L8) to inhibit lactic acid export while enhancing photodynamic therapy. L8BO has a long pi conjugated structure that effectively enhances its light capture performance; strong intramolecular charge transfer (ICT) characteristics make it have a small singlet-triplet energy gap (Delta E S-T ), so it can efficiently produce active free radicals; a longer alkane chain is also introduced as a spacer in the molecular plane skeleton to provide space for molecular movement and promote photothermal conversion. SY can specifically reduce the expression of monocarboxylate transporter 4 (MCT4), thereby blocking lactic acid export, inducing intracellular acidosis, inhibiting the formation of lactic acid-induced immunosuppressive microenvironment, and enhancing the effect of photodynamic therapy. The composition L8@SY nanoparticles (NPs) of the present application realizes the simultaneous generation of type I and type II reactive oxygen species (ROS), overcoming the limitations of oxygen-dependent type II photodynamic therapy, and the total active oxygen production is as high as 503 times. At the same time, L8@SY NPs also have a photothermal conversion efficiency (PCE) as high as 46.2%. Under the trigger of lactic acid export blocking-induced intracellular acidification, the L8@SY NPs of the present application can strongly activate the maturation of dendritic cells (DCs) and induce massive infiltration of T lymphocytes. At the same time, this therapy can also promote the differentiation of natural killer (NK) cells and memory T cells, thereby effectively inhibiting local and distant tumor progression and simultaneously suppressing lung metastasis.

[0017] Compared with the prior art, the present application has the following advantages:

[0018] (1) The present application co-delivers MCT4 inhibitor xilonixapam (SY) and photodiagnostic agent L8BO (L8), which on the one hand realizes excellent active oxygen generation and higher photothermal conversion efficiency, and on the other hand reshapes the immunosuppressive microenvironment by blocking lactic acid export, enhancing photodynamic-photothermal synergistic therapy of tumors.

[0019] (2) The present application uses glutathione (GSH) responsive nanocarriers to achieve specific release of SY, target inhibition of expression of monocarboxylate transporter 4 (MCT4), block lactic acid export to induce intracellular acidosis, reshape the immunosuppressive tumor microenvironment (ITM), and significantly amplify the anti-tumor performance of photodynamic therapy.

[0020] (3) The present application uses a metabolic-immune circuit reconnection strategy to promote antigen presentation, maturation of dendritic cells (DCs), and differentiation of effector T cells / NK cells. Strong immune response effectively inhibits the growth of primary tumors and distant tumors, and significantly reduces lung metastasis, providing a new perspective for tumor treatment.

[0021] (4) The L8@SY NPs of the present application exhibit excellent efficacy in in vivo experiments: nearly completely inhibit primary tumors (inhibition rate 99.6%), significantly inhibit distal tumors (inhibition rate 68.6%), and at the same time reduce lung metastasis. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Figure 1. (a) Preparation process of L8@SY NPs; (b) L8@SY NPs enhance phototherapy by blocking lactate efflux to induce tumor cell apoptosis and immunogenic death; (c) Reconnection of metabolic-immune circuit activates immune response and inhibits tumor growth and metastasis.

[0023] Figure 2 Figure 2. (a) is the molecular structure of L8; (b) and (c) are the front and side views of L8 in S0 state, respectively; (d) is the electrostatic potential distribution of L8; (e) is the frontier molecular orbital analysis; (f) and (g) are the absorption and emission spectra of DMSO solution of L8, L8 NPs and L8@SY NPs, respectively; (h) is the transmission electron microscopy image of L8@SY NPs, scale bar: 100 nm; (i) is the particle size distribution of L8@SY NPs and (j) is the stability.

[0024] Figure 3 Figure 3. (a), (b), (c) are the ROS generation capacity of DCFH alone, L8 NPs and L8@SY NPs, respectively; (d), (e) (f) are the identification of ROS types by DPBF, HPF and DHR123, respectively; (g) is the ESR signal intensity of different ROS under dark or laser irradiation; (h) is the photothermal heating curve of water, L8 NPs and L8@SY NPs (100 μg mL-1); (i) is the laser power dependence of L8@SY NPs; (j) is the concentration dependence of L8@SY NPs; (k) is the comparison of photothermal stability of L8@SY NPs and ICG. -1 ) of water, L8 NPs and L8@SY NPs (100 μg mL-1); (i) is the laser power dependence of L8@SY NPs; (j) is the concentration dependence of L8@SY NPs; (k) is the comparison of photothermal stability of L8@SY NPs and ICG.

[0025] Figure 4 Figure 4. (a) is the flow cytometry analysis of fluorescence intensity of EMT6 cells after co-incubation with L8@SY NPs; (b) and (c) are the cell survival rates of Beas-2b, EMT6 cells treated with different concentrations of L8@SY NPs in the dark (mean ± SD, n = 3); (d) is the survival rate of EMT6 cells incubated with L8 NPs or L8@SY NPs exposed to laser irradiation (mean ± SD, n = 3); (e) is the ROS generation, LPO, MMPs changes and live / dead staining of EMT6 cells by different treatments, scale bar: 100 μm; (f) is the apoptosis analysis by flow cytometry using different treatments.

[0026] Figure 5 Figure 8. (a) is the detection of intracellular pH changes in EMT6 cells after different treatments using pHrodo Green probe, scale bar: 100 pm; (b) is the fluorescence imaging of HMGB1 release or CRT exposure after different treatments, scale bar: 100 pm; (c) is the schematic diagram of DCs maturation induced by EMT6 cells after different treatments; (d) is the flow cytometry analysis of CD80 and CD86 expression in DCs cells.

[0027] Figure 6 Figure 9. (a) is the photograph of tumors collected from different treatment groups; (b), (c) are the volume changes of primary tumors and distant tumors, respectively, for various treatment modalities (I: PBS; II: PBS+L; III: L8 NPs; IV: L8 NPs+L; V: L8@SY NPs; VI: L8@SY NPs+L); (d), (e) are the weights of primary tumors and distant tumors, respectively, for different groups; (f) is the weight of spleen for different groups of mice; (g) is the body weight change of mice in different groups during treatment; (h) is the H&E, Ki67, TUNEL, HMGB1, CRT and CD8 + T cell staining, scale bar = 100 pm; data are presented as mean ± standard deviation (n = 5); P values are indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0028] Figure 7 Figure 10. Representative flow cytometry results and quantitative analysis of (a) mature dendritic cells, (b) CD8 + T cells, (c) CD44 + CD62 - memory T cells and (d) CD3 + CD161 + natural killer T cells for different groups (I: PBS; II: PBS+L; III: L8 NPs; IV: L8 NPs+L; V: L8@SY NPs; VI: L8@SY NPs+L); (e) is the H&E staining of whole lung for different groups of mice, scale bar: 2 mm; data are presented as mean ± standard deviation (n = 3). P values are indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. DETAILED DESCRIPTION

[0029] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the drawings.

[0030] Example 1

[0031] Preparation of L8@SY NPs, such as Figure 1 As shown, it includes the following steps:

[0032] A 1 mL tetrahydrofuran solution containing 5 mg L8, 2 mg Syrosingopine (SY), and 35 mg DSPE-SS-PEG2000 was added to 9 mL of deionized water and then sonicated at 50% power for 1 minute. The resulting nanoparticle solution was purified using an ultrafiltration tube with a molecular weight of 3000 to finally prepare L8@SY NPs.

[0033] The preparation of L8 NPs includes the following steps:

[0034] A 1 mL tetrahydrofuran solution containing 5 mg L8 and 25 mg DSPE-SS-PEG2000 was added to 9 mL of deionized water, followed by sonication at 50% power for 1 minute. The resulting nanoparticle solution was purified using an ultrafiltration tube with a molecular weight of 3000 to finally prepare L8 NPs.

[0035] Performance testing:

[0036] 1. Basic physical and chemical properties of materials

[0037] L8 employs a symmetrical electron acceptor-donor-acceptor (ADA) structure, where its double electron-withdrawing ends and electron-rich core form a highly efficient charge transfer channel, such as... Figure 2 (a) This ADA structure not only reduces the singlet-triplet bandgap (ΔE) S-T This also improves intersystem crossing (ISC) efficiency and enhances the generation of reactive oxygen species (ROS). The planar π-conjugated system endows L8 with high light absorption capacity, but simultaneously introduces aggregation-induced fluorescence quenching (ACQ). To address this issue, this invention introduces branched alkyl groups as spacer units into the molecule. This alleviates the ACQ effect and provides space for molecular motion, promoting free molecular rotation to enhance photothermal conversion performance. Density functional theory (DFT) calculations show that the introduction of alkyl chains leads to dihedral distortion but does not affect its core planarity, such as... Figure 2 In (b) and (c), the electrostatic potential (ESP) distribution plots show a localized electron-deficient region at the cyano substituent, in stark contrast to the electron-rich region on the conjugated backbone. This is consistent with the designed charge distribution pattern, such as... Figure 2 (d) Frontier orbital analysis shows that the highest occupied molecular orbitals (HOMOs) are distributed in the π-conjugated framework, while the lowest unoccupied molecular orbitals (LUMOs) are concentrated at the electron-deficient ends. The 2.03 eV band gap can provide a driving force for charge separation, such as... Figure 2 Middle (e).

[0038] The present application takes DSPE-SS-PEG2000 as a carrier, and prepares L8@SY NPs by nano-precipitation method. The absorption peak is red-shifted from 710 nm in DMSO to 812 nm, indicating the formation of J-aggregates, as shown in Figure 2 Fig. (f). The absorption peak of L8@SY NPs is red-shifted from 710 nm to 812 nm, which is conducive to the irradiation of 808 nm laser, and the red-shift of the emission peak is conducive to biological imaging. The photoluminescence (PL) spectrum also reflects this trend, with the emission peak shifted from 781 nm to 836 nm Figure 2 Fig. (g). Transmission electron microscopy (TEM) shows that the nanoparticles are spherical particles with a particle size of about 310 nm, as shown in Figure 2 Fig. (i); and can exist stably in water for 14 days, as shown in Figure 2 Fig. (j).

[0039] 2. ROS generation and photothermal conversion performance

[0040] The present application quantitatively evaluates the photosensitivity of L8 NPs and L8@SY NPs by using 2',7'-dichlorodihydrofluorescein (DCFH) probe. Under 808 nm laser irradiation, the DCFH control group only shows weak fluorescence, while the fluorescence signal of L8 NPs and L8@SY NPs at 525 nm is enhanced by 350 times and 503 times respectively, as shown in Figure 3 Figs. (a)-(c). Through detection by different specific probes (DPBF, HPF, DHR123), it is found that L8@SY NPs can decompose 37.21% of DPBF to generate 1O2 after 5 minutes of irradiation, and can also effectively generate ·OH and O2 - ·, as shown in Figure 3 Figs. (d)-(f). Electron spin resonance (ESR) spectrum further confirms that L8@SY NPs can simultaneously generate type I and type II reactive oxygen species, as shown in Figure 3 Fig. (g).

[0041] Photothermal performance test shows that after 808 nm laser irradiation at a power density of 0.6 W cm -2 for 5 minutes, the temperature of L8 NPs and L8@SY NPs solution rises to 66.3℃ and 64.2℃ respectively, as shown in Figure 3 Fig. (h). The temperature rise shows a good linear relationship with the laser power (0.2-0.6 W cm -2 ), nanoparticle concentration (25-100 μg mL -1 ), as shown in Figure 3 Figs. (i)-(j). After three laser cycles, L8@SY NPs show better photothermal stability than ICG Figure 3medium (k) with a high photo-thermal conversion efficiency of 46.2%. Therefore, L8@SY NPs is a dual-modal nano-platform with PDT and PTT potential.

[0042] 3. In vitro anti-tumor effect

[0043] The internalization kinetics of L8@SY NPs in EMT6 cells was first systematically evaluated by flow cytometry, and the results showed that the intracellular red fluorescence intensity was significantly enhanced with the prolongation of incubation time, from 0.33% at the initial 0 hour to 99.2% at 12 hours, thus determining 12 hours as the optimal cell uptake time, as shown in Fig. 2(a). Figure 4 CCK-8 cytotoxicity experiments showed that L8@SY NPs exhibited good biocompatibility (survival rate 93.7%) to normal Beas-2b cells, while showing selective toxicity (survival rate 72.2%) to EMT6 tumor cells, and this difference was mainly due to its specific response ability to overexpress GSH in the tumor microenvironment, as shown in Fig. 2(b) and (c). Figure 4 Under 808 nm laser irradiation, L8@SY NPs exhibited a significant concentration-dependent cell killing effect, and in the highest concentration treatment group, L8 NPs and L8@SY NPs inhibited the survival rate of EMT6 cells to 7.2% and 4.1%, respectively, as shown in Fig. 2(d). Figure 4

[0044] By detecting the intracellular ROS level with DCFH probe, it was found that compared with the PBS control group and the L8 NPs treatment group, L8@SY NPs produced the most significant green fluorescence signal after laser excitation, confirming its optimal ROS generation ability, as shown in Fig. 2(e). Figure 4 Further mechanism studies showed that L8@SY NPs not only could induce the basic level of lipid peroxidation by consuming intracellular GSH, but also could trigger significant redox imbalance under laser triggering; JC-1 staining results showed that the L8 NPs and L8@SY NPs groups treated with laser presented progressive green fluorescence, indicating the gradual decline of mitochondrial membrane potential (MMP); flow cytometry quantitative analysis showed that the cell survival rate of the L8@SY NPs treatment group activated by laser was the lowest (6.5%), and these results collectively proved the excellent performance of L8@SY NPs as a light-activated anti-tumor agent, as shown in Fig. 2(f). Figure 4

[0045] 4. Evaluation of activated immune response

[0046] ​​To clarify the mechanism of L8@SY NPs improving the tumor immunosuppressive microenvironment by regulating lactate metabolism, the inventors first detected the expression level of lactate transporter MCT4. The results showed that the basic MCT4 expression of EMT6 cells was high, and after L8@SY NPs combined with laser irradiation treatment, the MCT4 expression was significantly down-regulated by 87%, proving that SY can effectively inhibit the activity of MCT4. It was found by detecting with pHrodo Green fluorescent probe that the intracellular pH value of the L8@SY NPs treatment group was significantly reduced, such as Figure 5 Fig. 3(a), which is directly related to the blockage of MCT4-mediated lactate efflux.

[0047] Further study on the effect of L8@SY NPs inducing immunogenic cell death (ICD), the inventors focused on detecting the changes of two key damage-associated molecular pattern (DAMP) molecules: nuclear translocation of high mobility group protein B1 (HMGB1) and membrane surface exposure of calreticulin (CRT). Immunofluorescence results showed that HMGB1 in the control group and the simple NPs treatment group was mainly located in the nucleus (red fluorescence); after laser irradiation, HMGB1 nuclear translocation phenomenon occurred in the L8 NPs group, while the L8@SY NPs group showed the most significant cytoplasmic distribution of HMGB1, such as Figure 5 Fig. 3(b). At the same time, L8@SY NPs combined with laser treatment can induce a large amount of CRT to accumulate on the cell surface, confirming that it can effectively trigger DAMP release. To systematically evaluate the immune activation effect, the inventors detected the promoting maturation effect of tumor cell supernatant of different treatment methods on DC2.4 cells. Flow cytometry analysis showed that in the L8@SY NPs combined with laser treatment group, the expression level of co-stimulatory molecules CD80 / CD86 on the surface of DC2.4 cells was significantly up-regulated by 37.5% compared with the control group, such as Figure 5 Fig. 3(c) and (d). These results fully prove that L8@SY NPs can not only directly kill tumor cells through photodynamic / photothermal effect, but also promote the maturation of dendritic cells by inducing ICD, thereby activating systemic anti-tumor immune response.

[0048] 5. In vivo anti-tumor photoimmunotherapy

[0049] The inventors determined that 12 hours after injecting the drug was the best treatment time through photoacoustic imaging and small animal fluorescence imaging. Through the evaluation of 15-day observational study, it was found that the tumor in the non-irradiation group continued to grow, indicating that the efficacy of simple NPs was limited; while the tumor growth inhibition rate of the L8@SY NPs combined with laser irradiation group reached 99.6%, confirming that blocking lactate efflux can enhance the effect of photoimmunotherapy, such as Figure 6 Fig. 4(b). The growth trend of distant tumors is also consistent with this, such as Figure 6 Fig. 4(c).

[0050] Hematoxylin-eosin (H&E) staining of tumor specimens showed that the L8@SY NPs+L group had extensive necrotic areas, in sharp contrast to the intact structure of the control group, as shown in Figure 6 Fig. 16A. Ki67 staining indicated that the L8@SY NPs+L group had significantly fewer proliferating cells, and TUNEL detection showed significantly more apoptotic cells. Immunofluorescence detection confirmed that the L8@SY NPs+L group had the highest levels of HMGB1 and CRT, and the CD8 + T cell infiltration increased, indicating that this treatment could effectively induce ICD and activate an anti-tumor immune response.

[0051] 6. Evaluation of the remodeling immune microenvironment

[0052] Flow cytometry showed that the L8@SY NPs triggered an immune-stimulating cascade under laser irradiation: in the tumor-draining lymph nodes, the mature DC cells (CD80 + CD86 + ) accounted for 46.0%, which was 2.65 times higher than in the other groups (27.9%-41.7%), confirming that the inhibition of lactate efflux enhanced the activation of antigen-presenting cells, as shown in Figure 7 Fig. 16A(a). While DC maturation is directly related to the expansion of cytotoxic T cells, the inventors found that the L8@SY NPs+L group had higher CD3 + CD8 + T cell infiltration rates in the primary tumors, which were 2.29, 2.17, 2.01, 1.30, and 1.45 times higher than in the other groups, as shown in Figure 7 Fig. 16A(b). Longitudinal monitoring showed that the L8@SY NPs+L group had 21.7% memory T cells (CD8 + CD62L - CD44 + ) in the spleen, which was much higher than in the other groups, indicating that it could induce long-term immune memory, as shown in Figure 7 Fig. 16A(c). NK cells (CD3 + , CD161 + ) have both innate and adaptive immune functions, Figure 7 Fig. 16A(d) showed that the L8@SY NPs+L group had more NK T cells in the primary tumors. In summary, the L8@SY NPs+L could remarkably induce ICD, enhance DC maturation, activate CD8 + T cells, expand memory T cells, increase natural killer T cell infiltration, produce strong whole-body anti-tumor immunity, and lung H&E staining showed fewer metastatic nodules, as shown in Figure 7 Fig. 16A(e), confirming that it could inhibit tumor growth and metastasis.

[0053] In summary, compared with L8 NPs, the synergistic effect of L8 and SY in L8@SY NPs not only maintains excellent reactive oxygen species generation and higher photothermal conversion efficiency, but also SY in the composition can remodel the immune microenvironment by blocking lactate efflux, activate the expression of various immune cells, and further inhibit the growth of distant tumors and lung metastasis.

Claims

1. A composition for blocking lactate efflux and enhancing tumor photoimmunotherapy, characterized in that, Including cilosporin and phototherapy agent L8BO.

2. The composition for blocking lactate efflux and enhancing tumor photoimmunotherapy according to claim 1, characterized in that, The mass ratio of the added cilosporin and phototherapy agent L8BO is 1:(1-5).

3. The method for preparing nanoparticles of the composition for blocking lactate efflux and enhancing tumor photoimmunotherapy according to claim 1, characterized in that, Includes the following steps: (1) Add cilosporin and phototherapy agent L8BO and DSPE-SS-PEG2000 to tetrahydrofuran solution; (2) Add deionized water and then sonicate for 1-30 minutes.

4. The preparation method according to claim 4, characterized in that, The volume ratio of the added tetrahydrofuran solution to deionized water was 1:

9.

5. The application of the composition for blocking lactate efflux and enhancing tumor photoimmunotherapy according to claim 1, characterized in that, This is used to prepare photodynamic-photothermal synergistic therapeutic agents that block lactate efflux.

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