A nano-drug co-loading photothermal therapeutic agent and tlr agonist and preparation and application thereof

Abstract

The application belongs to the technical field of biological medicine, and relates to a strategy of immunotherapy combined with photothermal therapy (PTT), and specifically discloses a nano drug co-loading a photothermal therapeutic agent and a TLR agonist and preparation and application. Amphiphilic block polymers, a TLR agonist and a photothermal therapeutic agent are mixed in a solution to obtain the nano drug co-loading the photothermal therapeutic agent and the TLR agonist. The application adopts an integrated vesicle nano drug (t-PAu / PIC) co-loading a photothermal therapeutic agent (such as AuNC) and a TLR agonist (such as PIC), and implements efficient combination of PTT and immunotherapy. Results show that only one intratumoral injection and one NIR light irradiation can achieve efficient tumor inhibition and generate strong and persistent immune memory; after t-PAu / PIC is combined with CTLA4 antibodies, 100% of mice can be cured.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to a strategy of immunotherapy combined with photothermal therapy (PTT), and specifically discloses a nano drug co-loading a photothermal therapeutic agent and a TLR agonist and preparation and application thereof. BACKGROUND

[0002] Emerging immunotherapy shows great potential by stimulating or regulating the patient's own immune system to eliminate tumor cells, but the proportion of patients benefiting from the current clinical treatment is still low (the response rate of lung cancer patients is only about 25%), especially the cold tumors with relatively low immunogenicity (such as the response rates of triple-negative breast cancer and malignant brain glioma patients are only <20% and <10% respectively). It is imperative to explore new and more effective therapies. The photosensitizer of photothermal therapy (PTT) can convert absorbed light into heat, causing irreversible damage to tumor tissue by heating. PTT has the advantages of non-invasiveness and high specificity in treating tumors, and has a significant effect on primary solid tumors, but its clinical promotion also faces obvious obstacles, such as the limited depth of laser penetration, almost no effect on tumors or metastases outside the irradiation area, and even leading to tumor recurrence and metastasis; high temperature of PTT leads to up-regulation of heat shock proteins in tumor cells, inducing heat resistance; high temperature can also induce tumor cells to produce immunosuppressive cytokines, causing tumor immune escape, metastasis and invasion. In addition, PTT can induce tumor cell apoptosis or immunogenic death within a certain temperature range, and the released DAMP can stimulate immune cells as new antigens, enhancing the immunogenicity of tumors, but the immune response induced by PTT alone is weak and cannot be sustained. SUMMARY

[0003] The present application adopts an integrated vesicle nano drug (t-PAu / PIC) co-loading a photothermal therapeutic agent (such as gold nanoclusters AuNC) and a TLR agonist (such as poly(I:C)), and implements efficient combination of PTT and immunotherapy. The results show that only one intratumoral injection and one NIR light irradiation can achieve efficient tumor inhibition and produce strong and persistent immune memory; 100% of mice can be cured after t-PAu / PIC combined with CTLA4 antibody.

[0004] The present application adopts the following technical solutions:

[0005] A nano drug co-loading a photothermal therapeutic agent and a TLR agonist, comprising a reduction-sensitive disulfide cross-linked vesicle and a TLR agonist and a photothermal therapeutic agent loaded therein, wherein the TLR comprises one or more of TLR3, TLR7 / 8 and TLR9. Preferably, the TLR agonist comprises poly(I:C); and the photothermal therapeutic agent comprises a gold nanomaterial, such as gold nanoclusters. The TLR agonist and the photothermal therapeutic agent of the present application are located together in the reduction-sensitive disulfide cross-linked vesicle.

[0006] The application discloses a preparation method of the nanomedicine co-loading the photothermal therapeutic agent and the TLR agonist, and comprises the following steps: mixing amphiphilic block polymers, a TLR agonist and a photothermal therapeutic agent in a solution to obtain the nanomedicine co-loading the photothermal therapeutic agent and the TLR agonist. Specifically, the solution of the amphiphilic block polymers is added into a mixture of the TLR agonist, the photothermal therapeutic agent and a buffer solution to obtain the nanomedicine co-loading the photothermal therapeutic agent and the TLR agonist; preferably, the mixture is placed and dialyzed to obtain the nanomedicine co-loading the photothermal therapeutic agent and the TLR agonist. The amphiphilic block polymers form reduction-sensitive double-sulfur cross-linked vesicles for loading the TLR agonist and the photothermal therapeutic agent.

[0007] In the application, the mass ratio of the TLR agonist to the photothermal therapeutic agent is 1: (0.1-50), preferably 1: (0.1-30), more preferably 1: (0.1-20), and further preferably 1: (0.2-10).

[0008] In the application, the amphiphilic block polymer is a hydrophilic segment-P (B-DTC)-cationic segment, or the amphiphilic block polymer is a hydrophilic segment-P (B-DTC)-cationic segment and A-hydrophilic segment-P (B-DTC); wherein A is a targeting molecule, preferably an integrin targeting molecule, such as cRGD; B is a cyclic ester or a cyclic carbonate monomer unit, such as trimethylene carbonate monomer TMC, lactide monomer LA, caprolactone monomer CL, etc.; DTC is dithiacyclopentane trimethylene carbonate; and the cationic segment is one of spermine and small molecular weight PEI, preferably spermine. As an example, the amphiphilic block polymer is preferably PEG-P (TMC-DTC)-Sp, or PEG-P (TMC-DTC)-Sp and cRGD-PEG-P (TMC-DTC).

[0009] Correspondingly, when the amphiphilic block polymer is a hydrophilic segment-P (B-DTC)-cationic segment, a non-targeted reduction-sensitive double-sulfur cross-linked vesicle is formed; when the amphiphilic block polymer is a hydrophilic segment-P (B-DTC)-cationic segment and A-hydrophilic segment-P (B-DTC), a targeted reduction-sensitive double-sulfur cross-linked vesicle is formed. Preferably, the molar percentage of A-hydrophilic segment-P (B-DTC) in the polymer is 0-40%, excluding 0; further preferably, the molar percentage of A-hydrophilic segment-P (B-DTC) in the polymer mixture is 2-30%, and more preferably the molar percentage is 10-25%.

[0010] The application discloses an application of the photothermal therapeutic agent and the TLR agonist in the preparation of a compound nanomedicine. The compound nanomedicine refers to the photothermal therapeutic agent and the TLR agonist co-loaded in a vesicle.

[0011] The application discloses a combined medicine, which comprises the nano-medicine co-loading the photothermal therapeutic agent and the TLR agonist and other anti-tumor medicines.

[0012] The application discloses application of the nano-medicine co-loading the photothermal therapeutic agent and the TLR agonist or the combined medicine in preparation of an anti-tumor medicine.

[0013] In the application, the anti-tumor medicine can take the nano-medicine co-loading the photothermal therapeutic agent and the TLR agonist as a main active ingredient, can take the nano-medicine co-loading the photothermal therapeutic agent and the TLR agonist and other medicines as active ingredients, or can be combined with other treatment methods, such as radiotherapy and thermotherapy.

[0014] The application is based on a reduction-sensitive double-sulfur cross-linked polymer vesicle of PEG-P(TMC-DTC)-sp, a vesicle nano-medicine (t-PAu / PIC) co-loading a photosensitizer gold nanocluster (AuNC) and poly(I:C) is designed and prepared, and the vesicle nano-medicine is used for photothermal immunotherapy of tumors and combined immunotherapy with ICB. The AuNC in the vesicle membrane is derived from a commercial CT contrast agent AuroVist, which is a nanocluster with a core of about 1.9 nm, is an ideal PTT photosensitizer, but has a small particle size and is easily removed quickly, and poor retention is a defect, so far, there is no related report on using the AuNC for PTT. The AuNC is co-loaded into the vesicle to change the biological distribution of the AuNC, and the combination of PTT and t-PPIC is expected to organically combine the non-invasive heating TME of PTT, increase the tumor infiltration of CTL and the strong adjuvant effect of t-PPIC, and achieve the purposes of eradicating tumors and improving the cure rate. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1Physicochemical properties of t-PAu / PIC. (A) Empty vesicles Ps, single-loaded AuNCs and co-loaded Au / PIC vesicles, (B) Particle size and size distribution of t-PAu / PIC with different AuNC loadings (PIC concentration: 500 pg / mL). (C) Visible light absorption spectra of AuNCs and PAu at different concentrations. TEM images of t-PAu / PIC obtained after (D) and (E) 10 mM GSH pretreatment of AuNCs during preparation. (F) Photothermal effect of AuNC solutions at different concentrations and (G) PAu and AuNC + Ps mixture. (H) Photothermal effect and photothermal stability of PAu / PIC after three cycles of NIR irradiation. (I) Particle size and size distribution of t-PAu / PIC under different treatment conditions. All “L” in the figures indicates NIR irradiation, 808 nm, 2 W / cm 2 . AuNC concentration: 200 pg / mL, unless otherwise specified.

[0016] Figure 2 Effects of t-PAu / PIC formulation on LLC cells and BMDCs. (A) Survival rate of LLC cells treated with t-PAu / PIC plus NIR irradiation (2 W / cm 2 , 5 min, 24 h, n = 5, PIC conc: 1 pg / mL). (B) Toxicity of t-PPIC at different concentrations on LLC cells (24 h, n = 6). (C) Apoptosis and CRT expression (D) and ATP secretion (E) of LLC cells induced by t-PAu / PIC plus NIR irradiation (1 W / cm 2 , 5 min). For C-E, the medium was replaced with fresh medium after 4 h of loading, followed by NIR irradiation and incubation for another 20 h (n = 3, AuNC conc: 300 pg / mL, PIC conc: 10 pg / mL; L: 2 W / cm 2 , 5 min). (F) Maturation of BMDCs induced by t-PAu / PIC plus NIR treatment of LLC cells (n = 3, AuNC conc: 200 pg / mL, PIC conc: 5 pg / mL; L: 1 W / cm 2 , 5 min).

[0017] Figure 3 Effects of t-PAu / PIC, PAu / PIC and AuNC (i.t., L: 808 nm, 2 W / cm 2Therapy of LLC mice with t-PAu / PIC (i.t., L: 808 nm, 2 W / cm2, 5 min) or in combination with CTLA-4 antibody (Ab, i.v., 1.0 mpk) (n = 6). (A) Workflow. (B) Tumor growth, (C) individual tumor growth, (D) body weight and (E) survival curves of mice.

[0018] Figure 4 Immunological analysis of LLC mice treated with t-PAu / PIC + L + Ab (n = 5). (A) Workflow. Tumor weight of each group (B). The content of (C) CD80 + DCs, (D) NKG2D + NK cells, (E) CD3 + CD8 + T cells (CTL), (F) CD25 + CTL, (G) IFN-γ + CTL, (H) CD3 + CD4 + T cells, (I) Treg proportion of CD4 + T cells, (J) CTL / Treg and (K) M1M / M2M ratio and (L) MDSCs infiltration. The content of (M) mDCs and (N) MHC II + mDCs in lymph nodes. The concentration of (O) NKG2D + NK cells, (P) IL-6 and (Q) IFN-β in peripheral blood. The content of (R) CD3 + CD8 + T cells, (S) CD25 + CTL, (T) CD3 + CD4 + T cells, (U) CTL / Treg ratio and (V) MDSCs content.

[0019] Figure 5 Therapy of LLC mice with t-PAu / PIC (i.t., L: 808 nm, 2 W / cm 2 , 5 min) or in combination with CTLA-4 antibody (Ab, i.v., 1.0 mpk) (n = 4). (A) Workflow. (B) Tumor growth curve, (B) individual tumor growth curve, (D) body weight and (E) survival curves of mice.

[0020] Figure 6 Memory effect study of t-PAu / PIC photothermal immunotherapy (n = 3). As Figure 5Tumor growth curves of the re-challenged mice (A) at day 90 after re-inoculation of LLC cells; CD4 + and CD8 + memory T cells in the peripheral blood (C) and spleen (D) of the mice at 60 days after re-inoculation (day 150) (n = 2 or 3).

[0021] Figure 7 As shown in FIG. 1A, the tumor growth curves of the re-challenged mice (A) at day 90 after re-inoculation of LLC cells; and the CD4 Figure 3

[0022] Figure 8 As shown in FIG. 1A, the tumor growth curves of the re-challenged mice (A) at day 90 after re-inoculation of LLC cells; and the CD4 Figure 5

[0023] Figure 9 As shown in FIG. 2A, the work flow of the photothermal immunotherapy of t-PAu / PIC (i.t., L: 808 nm, 2 W / cm 2 , 5 min) or combined with CTLA-4 antibody (Ab, i.v., 1.0 mpk) on triple-negative breast cancer 4T1-luc mice (n = 6). (A) Experimental schedule. The tumor growth (B), individual tumor growth (CR: cured), (C) body weight, and (D) survival curves of the mice.

[0024] Figure 10 As shown in FIG. 2A, the work flow of the photothermal immunotherapy of t-PAu / PIC (i.t., L: 808 nm, 2 W / cm 2 , 5 min) or combined with CTLA-4 antibody (Ab, i.v., 1.0 mpk) on triple-negative breast cancer 4T1-luc mice (n = 6). (A) Experimental schedule. The tumor growth (B), individual tumor growth (CR: cured), (C) body weight, and (D) survival curves of the mice.

[0025] Figure 11 As shown in FIG. 2A, the work flow of the photothermal immunotherapy of t-PAu / PIC (i.t., L: 808 nm, 2 W / cm DETAILED DESCRIPTION ​​

[0026] As an example, the present application discloses a nanomedicine co-loading gold nanoclusters and TLR3 agonist, which can induce strong and persistent anti-tumor photothermal immunotherapy. The immune cells rich in the tumor margin can diffuse and penetrate deeper into the tumor with the expansion of blood vessels and the disintegration of the tumor interstitial layer caused by photothermal effect, while the tumor-associated antigens need the assistance of immune adjuvants to induce strong anti-tumor immune response of the diffusing immune cells. The combination of PTT and immunotherapy is very beneficial to tumor treatment.

[0027] The technical progress of the present application is illustrated below by specific experiments, and the raw materials used are existing products. The specific preparation operation and performance test are conventional techniques.

[0028] AuroVist (AuNC, 1.9 nm, Nanoprobes, in solution form), polyinosinic acid (poly (I:C), LMW, 0.2-1 kb, InvivoGen, referred to as PIC) and other reagents and kits are directly used after purchase. PEG-P (TMC-DTC), cRGD-PEG-P (TMC-DTC) and PEG-P (MC-DTC)-sp are all synthesized according to existing methods. Mouse fluorescently labeled antibodies for flow cytometry analysis of immune memory cells are all purchased from Biolegend. For dead and live staining: APC / Cy7-Zombie NIR Fixable Viability Kit. Blocking antibody: anti-CD16 / 32-TruStain FcX. Labeling immune cells: anti-CD45-Per / Cy5.5. Labeling T cells: anti-CD3-APC, anti-CD4-PE / Cy7, anti-CD8a-PE / Cy7. Labeling T CM : anti-CD62L-PE and T EM : anti-CD44-FITC. Near-infrared laser (FC808LNL-2W-FC) is used to provide 808 nm laser irradiation to mice. Thermocouple temperature meter (TA612C) is used to test the temperature of the laser irradiation area. The micro-morphology of the vesicles is determined by transmission electron microscopy (TEM, HT7700) at an acceleration voltage of 120 kV. The concentration of poly (I:C) (PIC), drug loading capacity (DLC) and encapsulation efficiency (DLE) are determined by NanoDrop 2000. Gold elements are quantified by inductively coupled plasma emission spectroscopy (ICP-OES) test. The absorption of gold nanocluster solution in the visible-near infrared wavelength range is determined by ultraviolet-visible spectrophotometer (UH5300). The instruments and test methods are conventional techniques.

[0029] Mouse non-small cell lung cancer LLC cells and mouse triple-negative breast cancer cells 4T1-luc were purchased from the China Academy of Sciences Cell Bank. LLC cells were cultured in DMEM medium (Gibco) containing 10% FBS, 1% penicillin-streptomycin (Genview Biotech); 4T1-luc was cultured in RPMI-1640 medium (Gibco) containing 10% FBS, 1% penicillin-streptomycin. BMDCs were obtained from the femur and tibia of C57BL / 6J female mice according to the existing method. All cells were cultured in monolayer at 37 ℃, 5% CO2 in a 3111 type incubator (Thermo, USA). C57BL / 6J female mice (6-8 weeks old, 18-20 g) and BALB / c female mice (6-8 weeks old, 18-20 g) were purchased from Vantianlihua Company and were strictly bred according to the regulations in a room with 25 ℃, 55% humidity and programmed light and dark. All animal experiments were approved by the Suzhou University Animal Care and Use Committee, and all experimental protocols complied with the Guide for the Care and Use of Laboratory Animals.

[0030] All data in the experiments of the present application were presented as mean ± standard deviation (SD), and the differences between groups were evaluated by ANOVA one-way variance analysis, *p < 0.05 was considered to have significant difference, **p < 0.01, ***p < 0.001 and ****p < 0.0001 were considered to have high significant difference.

[0031] Example 1 Preparation and characterization of PAu / PIC

[0032] The polymer vesicles co-loading gold nanoclusters and TLR3 agonists are prepared based on two copolymers of polyethylene glycol- b -poly (trimethylene carbonate- co -dithiacyclohexanetriylmethyl carbonate) - spermine (PEG-P(TMC-DTC)-sp, 5.0-(14-1.9)-0.2 kg / mol) and cRGD modified polymer cRGD-PEG-P(TMC-DTC) (0.4-7.5-(14-1.9) kg / mol). PEG-P(TMC-DTC)-sp is obtained by amidation reaction of CDI activated PEG-P(TMC-DTC) terminal hydroxyl group and spermine. The molecular weight of PEG-P(TMC-DTC)-sp is determined by GPC. 1The integration ratio of CDI (δ 8.1-8.2), spermine (δ 2.56-2.86) and PEG (δ 3.38, 3.65) in H NMR (600 MHz, CDCl3) spectrum can be used to calculate the functional degree of CDI and sp on the polymer, which are close to 100% and 92%, respectively. cRGD-P(TMC-DTC) is obtained by Michael addition reaction of NHS-PEG-P(TMC-DTC) and cRGDfC. The characteristic peak of NHS (δ 6.80) disappears in the NMR spectrum of the product due to the coupling of cRGD, indicating that the functionalization degree of cRGD is close to 100%. The synthesis and characterization methods of PEG-P(TMC-DTC)-sp, cRGD-PEG-P(TMC-DTC) and Cy5-labeled PEG-P(TMC-DTC) are the same as the prior art, and it has been proved that these polymers can form polymer vesicles.

[0033] The DMF stock solution (50 mg / mL) of PEG-P(TMC-DTC)-sp and the aqueous solution of PIC stock solution (5 mg / mL) are prepared for use. The commercially available AuNC solution is mixed with 10 mM GSH solution (1 / 1, v / v) and magnetically stirred (800 rpm) for 3 min. When the theoretical drug loading of AuNC is 2 wt. %~15 wt. %When the theoretical drug loading of AuNC is 2

[0034] The present application can co-load AuNC and poly(I:C) into cRGD-modified double-sulfur cross-linked polymer vesicles to obtain t-PAu / PIC by the above simple method. Specifically, cRGD-P(TMC-DTC) and PEG-P(TMC-DTC)-sp are mixed according to the molar ratio of 20 / 80, and then self-assembled in the buffer containing 10 wt.% PIC (500 μg / mL) and AuNC treated with different concentrations of GSH.

[0035] The vesicles PAu and t-PAu with single AuNCs were prepared in the same way, but the water phase only contained AuNCs without PIC solution. Correspondingly, the vesicles PPIC and t-PPIC with single poly(I:C) were prepared in the same way, but the water phase only contained PIC solution without AuNCs. The empty vesicles Ps were obtained without adding AuNCs and poly(I:C).

[0036] The present application can finally obtain the co-loaded vesicles t-PAu / PIC with stable gold content of 16 wt.%, loading efficiency of more than 86%-100%, PIC content of up to 18.2 wt.%, and loading efficiency of 86%-97% by using a simple method (Table 1). The average particle size of the obtained vesicle nanodrugs is 54.6-58.6 nm, the particle size distribution is narrow (PDI: 0.07-0.2), and the particle size is very close to that of the two single-loaded vesicles Figure 1 The results of multiple independent preparations are similar, indicating that the preparation process has high repeatability (Table 1). In practical applications, the drug loading amount of Au and PIC and the polymer vesicle concentration can be reasonably regulated according to the temperature required for photothermal therapy of tumors, the wavelength and power density of near-infrared laser, and the amount of PIC adjuvant required for antitumor immunity in vivo, to achieve the best effect.

[0037] Table 1 Characterization of t-PAu / PIC a (n = 3)

[0038]

[0039] a Determined by ICP-OES; b Determined by NanoDrop; c Determined by DLS in HEPES (pH 6.8, 5 mM); d Polymersome conc. was 10 mg / mL, while in other cases it was 5 mg / mL.

[0040] The present application prepares integrin-targeted vesicles co-loaded with poly (I:C) (PIC) and gold nanoclusters (t-PAu / PIC) for efficient photothermal immunotherapy. AuNCs have collective plasmonic resonance coupling inside under laser irradiation, thus having the characteristics of resonance wavelength in the biological window and high photothermal conversion efficiency (up to 84%). Moreover, AuNCs are a commercial contrast agent for Micro-CT, which has low toxicity (LD 50 >1.4 g Au / kg), low viscosity, good water solubility (1.5 g Au / cc), but has the characteristic of fast blood clearance, which decreases to less than 15% within 5 min after injection and is almost completely excreted through the kidneys within 30 min. Therefore, there is currently no related report on the application of AuNCs as photosensitizers for PTT treatment.

[0041] To study the difference in visible light absorption curves of AuNCs before and after loading into vesicles, the absorbance curves of PAu and AuNC solutions with concentrations of 200 μg / mL to 1 mg / mL at wavelengths of 400 nm-1200 nm were measured by ultraviolet-visible spectrophotometry. AuNCs have high absorption whether loaded into vesicles or in solution, and have a wide absorption band in the visible light region to the infrared region rather than a characteristic absorption peak at a certain wavelength (the absorbance in the visible light region is higher than that in the infrared region). At the same gold concentration and the same wavelength, PAu has higher absorbance than AuNCs (Fig. 2C). Figure 1 This provides a basis for good PTT effect and possible near-infrared II region excitation.

[0042] The morphology of PAu prepared in two ways was determined by TEM. During the preparation of PAu, before adding the polymer solution to the aqueous phase, one way is to directly add AuNCs to the aqueous phase, and the other way is to first treat AuNCs with 10 mM GSH for 3 min and then add them to the aqueous phase. 10 μL of the two samples was taken onto a copper mesh carbon support film, and after standing for 20 min, the excess liquid was absorbed, and the TEM picture was taken after drying. If the purchased AuroVist solution is directly loaded without treatment, it cannot achieve the purpose of efficiently loading AuNCs into the vesicles. AuNCs are randomly distributed in the solution, and few enter the vesicles (Fig. 2D); while the AuroVist solution is treated with 10 mM GSH for 3 min before loading, AuNCs can be efficiently loaded into the hydrophobic membrane of the vesicles, and almost none are free outside the vesicles (Fig. 2E). Figure 1 Figure 1

[0043] To study the PTT effect of AuNCs, the photothermal conversion efficiency (η) of AuNCs was measured under near-infrared laser (L, 808 nm, 2 W / cm 2 ​​AuNC solution (concentration: 50, 100, 200 μg / mL) was added to each well of a 96-well plate at 200 μL, irradiated for 10 min, and the temperature of the solution was measured every minute during the irradiation process using a thermocouple thermometer. Figure 1 For temperature measurement, the probe was inserted into the solution without touching the container wall, and the probe was inserted for 5 s each time to stabilize the reading and avoid the influence of laser irradiation on the probe. To determine the difference in the photothermal effect of PAu, AuNC+empty carrier Ps, AuNC, and Ps (AuNC concentration: 200 μg / mL), the previous operation was irradiated (808 nm, 2 W / cm 2 ) for 5 min, and the temperature was measured for 5 min, and the remaining steps were the same as above. To study the thermal stability of PAu, PAu was subjected to three cycles of NIR irradiation and temperature measurement. After each irradiation (808 nm, 2 W / cm 2 ) for 5 min, it was stopped for 5 min. The solution temperature was continuously measured. The temperature rise of the PAu and AuNC solutions under near-infrared laser (NIR, L, 808 nm, 2 W / cm 2 , 10 min) irradiation was measured Figure 1 in the middle of G). PTT is usually designed in the near-infrared I region (650-850 nm) or II region (950-1350 nm) in the study, because light at these wavelengths can penetrate healthy tissue deeply to reach the tumor site and interact with the photosensitizer without causing damage to healthy tissue. The temperature rise of the two gold solutions at room temperature was concentration-dependent. The temperatures of the free AuNC solutions at 50, 100, and 200 μg / mL reached 47, 52, and 60°C, respectively, while the temperature of the PBS solution only rose from 25°C to 37°C, and all groups reached a plateau at about 5 min. When the AuNC concentration was the same at 200 μg / mL, under the same NIR irradiation, the temperature rise of the empty carrier Ps solution was comparable to that of the PBS group, and the temperature rise trend and the highest temperature reached by the PAu, free AuNC, and free AuNC+Ps mixture groups were basically the same, indicating that the AuNC loaded into the vesicle membrane completely retained the photothermal effect of AuNC and the thermal effect of PAu was recyclable and relatively stable Figure 1 in the middle of H). Here, the temperature rise of PAu and free AuNC is comparable to the thermal effect of various reported synthetic complex non-spherical gold nanoparticles (such as star-shaped, cage-shaped, spherical shell, and direct surface modification) or larger spherical gold nanoparticles at a similar gold concentration. PAu of the present application has similar and higher photothermal effects under simpler preparation operations, has advantages, and is conducive to production and application.

[0044] To investigate the effect of laser irradiation and heating on the integrity of PAu / PIC and drug release, the change of particle size and the leakage of poly(I:C) of PAu / PIC were studied under two ways of heating by laser irradiation and external heating. 500 μL of three groups of PAu / PIC were placed in dialysis bags with MWCO of 1000 kDa, and then the dialysis bags were placed in 1 mL of Hepes dialysis solution to avoid the influence of large concentration difference on drug leakage. After that, one group was at room temperature, the second group was irradiated by NIR for 5 min, and the third group was heated to about 60 ℃ for 5 min by a heating plate and the solution was slightly stirred. After 5 min of light irradiation, the sample was immediately taken out from the dialysis bag and the particle size and particle size distribution of the three groups of vesicles were tested by DLS, and the leakage of poly(I:C) in the solution outside the dialysis bag was tested by nanodrop. The change of particle size and the leakage of poly(I:C) of PAu / PIC under NIR light irradiation and external heating were determined. The DLS determination results showed that the particle size of PAu / PIC under the two heating ways was basically the same as that of the untreated PAu / PIC (Table 2). No leakage of PIC was detected in the dialysate after heating treatment (below the detection limit of Nanodrop) (Table 2), which verified the thermal stability of PAu / PIC. Therefore, t-PAu / PIC and PAu / PIC can exist in the form of vesicles after being delivered to tumor tissue and NIR light irradiation of AuNC to exert its photothermal effect, and can also play its function in tumor tissue: endocytosis by tumor cells and stimulation of immune cells, etc. Figure 1 Table 2 Determination of particle size and particle size distribution of t-PAu / PIC after PTT and the leakage of poly(I:C)

[0045] Table 2 Determination of particle size and particle size distribution of t-PAu / PIC after PTT and the leakage of poly(I:C)

[0046]

[0047] a Determined by DLS in Hepes (pH 6.8, 5 mM). b Determined by NanoDrop.L: 808 nm, 2 W / cm 2 , 5 min.

[0048] Example 2 Evaluation of the cell killing ability of PAu / PIC

[0049] The MTT method was used to study the toxicity of PAu / PIC and its combination with NIR light irradiation on LLC cells. The cells were plated in 96-well plates (5 x 10 3After overnight adhesion of the cells in 90 mL DMEM medium per well, the cells were divided into three groups. The first group received only 10 μL of PBS; the second group received 10 μL of PBS and PAu / PIC (PIC concentration: 1 μg / mL, Au concentration: 200 μg / mL); and the third group received laser irradiation (+L), i.e., the same PAu / PIC was added followed by NIR irradiation (808 nm, 2 W / cm²). 2 , 5 min); A parallel experiment was performed, adding 10 μL of free PIC, PPIC, and t-PPIC (PIC concentration: 0.01 to 100 μg / mL). After incubation for 24 h, 10 µL of MTT solution (5 mg / mL) was added and incubated at 37 ℃ for 4 h. The supernatant was removed, and 150 µL of DMSO was added and incubated at 37 ℃ on a shaker (100 rpm) for 10 min. The absorbance of the cells at 570 nm was measured using a microplate reader. Cell viability was the ratio of the absorbance of each sample to the absorbance of the PBS group (n = 4). The MTT assay results showed that under NIR illumination (808 nm, 2 W / cm²), the cells were viable. 2 At a gold concentration of 200 μg / mL or higher, PAu / PIC exhibited strong cytotoxicity against LLC cells, with a cell survival rate of only 25% (5 min). Figure 2 (A), while t-PPIC cannot directly kill LLC cells, and the concentration of PIC in all groups was within the range that would not harm the cells ( Figure 2 (Middle B). In contrast, unexposed cells showed no harm at any of the gold concentrations studied. The results confirm that high concentrations of PAu / PIC have good biosafety and can effectively kill tumor cells under NIR light irradiation.

[0050] Example 3: Assessment of PAu-induced apoptosis and immunogenic cell death (ICD) in LLC cells

[0051] LLC cells were fed at a rate of 3 × 10 4 The plates were seeded in 12-well plates and allowed to adhere overnight. The following groups were selected: PBS, t-PPIC, AuNC, PAu / PIC, t-PAu / PIC, L, AuNC+L, t-PAu+L, PAu / PIC+L, and t-PAu / PIC+L (Au concentration 300 μg / mL, PIC concentration 10 μg / mL, 1:30). After incubation for 4 h, the medium was changed. Then, each well in the "+L" group was exposed to NIR light (2 W / cm²). 2, 5 min). After another 20 h incubation, cells were digested with accutase, centrifuged (500 rpm, 8 min) and resuspended in PBS. Cells were processed according to the instructions of the double staining apoptosis kit and the ratio of early and late apoptotic cells was determined by FACS. The results showed that t-PAu+L, PAu / PIC+L and t-PAu / PIC+L could all induce more apoptosis in LLC cells than free AuNC+L, and t-PAu / PIC+L was the strongest. No significant cell apoptosis was observed when only the formulation or only the laser was added, indicating the important role of the nanoformulation in targeting tumor cells and the synergistic effect of PIC and PTT on cell toxicity. Figure 2 C).

[0052] Cell culture protocol and sample loading were the same as above. After 20 h incubation, the supernatant was taken to determine the concentration of ATP according to the kit instructions. Cells were trypsinized, washed twice with PBS; incubated with blocking antibody at 4 °C for 20 min; incubated with anti-CRT at room temperature for 1 h, washed twice with PBS; then incubated with Alexa Fluor 647 labeled goat anti-rabbit / mouse antibody at room temperature for 30 min, washed twice with PBS, and the content of CRT was determined by FACS. + The results showed that t-PAu+L, PAu / PIC+L and t-PAu / PIC+L could all induce LLC cells to produce higher concentrations of ATP and higher CRT expression, significantly higher than other groups, and t-PAu / PIC+L was the strongest. Figure 2 D, E).

[0053] To study the activation ability of LLC cells treated with t-PAu / PIC+L on BMDC, the tumor environment was simulated, and LLC cells were pre-plated in 12-well plates (1 x 10 5 / well, 900 mL of DMEM medium) overnight to adhere. 100 μL of t-PAu / PIC (PIC concentration: 5 μg / mL) was added, and the “+L” group was irradiated with a laser. After 20 h, 1 mL of BMDC (1 x 10 6FACS. Compared with L and PBS groups, t-PPIC had little effect on the activation of BMDCs at the concentration of 100 or 200 μg / mL AuNCs and 5 μg / mL PIC. Interestingly, the combination of NIR and t-PAu / PIC (t-PAu / PIC+L) greatly increased the proportion of mDCs at two concentrations of AuNCs, which demonstrated the potential of t-PAu / PIC+L photothermal immunotherapy for treating solid tumors Figure 2 F, ***p).

[0054] Different temperatures caused by PTT will have different therapeutic effects. Due to the penetration limit of NIR and heat diffusion, when the tumor is too large, even if the highest temperature in the irradiation area exceeds 72℃, the temperature of the tumor edge outside the effective light is lower than 42℃; while the thickness of the tumor tissue with temperature exceeding 43℃ is less than 6 mm. When PTT causes temperature higher than 60℃, proteins will denature, resulting in a greatly enhanced cell membrane permeability, and almost instantaneous tumor cell collapse and death. When PTT causes temperature to rise to the range of 42-46℃, the heat generated will cause irreversible damage to the microstructure of tumor cells, such as cell membrane, nucleus and mitochondria, which not only induces apoptosis and ICD, but also releases DAMP as TAA to enhance the immunogenicity of the tumor and increase the infiltration of CTL in TME, which is of great significance for combined immunotherapy to eradicate tumors, inhibit tumor recurrence and metastasis. According to the above experimental results, it can be inferred that for the case of insufficient temperature in the irradiation area and deeper area that may be encountered in practical application, t-PAu / PIC can efficiently kill cells under NIR irradiation, and also can induce ICD of tumor cells to produce highly immunogenic antigens, which demonstrates the necessity and feasibility of the photothermal immunotherapy of the present application.

[0055] Example Four Photothermal immunotherapy experiment and immune environment analysis of t-PAu / PIC for LLC lung cancer mice

[0056] To establish a mouse subcutaneous LLC lung cancer model, LLC cells (1×10 6 were resuspended in PBS containing 40% matrigel, and 50 μL / mouse was inoculated subcutaneously on the right hind leg of the mouse. About 10 days later, the tumor volume grew to 100-120 mm 3The experiment was started. To investigate the photothermal immunotherapy effect of intratumoral injection of PAu / PIC preparation plus NIR, the effects of AuNC loading, co-loading of AuNC and PIC, cRGD modification and combination with CTLA-4 monoclonal antibody (Ab) on the tumor inhibition ability of LLC mice were studied. When the tumor volume was 100-120 mm 3 When the tumor volume was 100-120 mm 2 , the mice were divided into 8 groups (n = 6), and were administered intratumorally (i.t.) on day 0. The photothermal group was additionally subjected to NIR irradiation (808 nm, 2 W / cm 3 , 5 min) once at 30 min after i.t. administration on the same day. The CTLA-4 antibody (Ab) group was administered on days 1, 4, and 7 (1 mpk, i.v.). The groups were specifically: PBS, PPIC, AuNC+PPIC+L, PAu+L, PAu / PIC+L, PAu / PIC+L+Ab, t-PAu / PIC+L, and t-PAu / PIC+L+Ab. The PIC dose was 1.25 mpk, and the AuNC dose was 0.5 mpk (1:0.4). The tumor volume, body weight, and state of the mice were monitored every 3 days, and the natural death or tumor volume greater than 2000 mm 2 was also determined as death, and the survival curve was plotted accordingly. The tumor volume was calculated by the formula V = 0.5 × L × W 6 , where L and W are the distances measured by a vernier caliper at the widest and narrowest parts of the tumor, respectively). On day 120, the cured mice in the PAu / PIC+L, PAu / PIC+L+Ab, t-PAu / PIC+L, and t-PAu / PIC+L+Ab groups were in good condition, and LLC cells were inoculated subcutaneously on the right hind leg above the previous inoculation site and in the same amount in some of the cured mice. GBM glioblastoma cells (1 × 10 3 / each, containing 25% matrigel) were inoculated subcutaneously on the right hind leg above the previous inoculation site in the other cured mice. The growth of the tumor, body weight, and state of the mice were monitored and recorded.

[0057] The anti-tumor activity of a single intratumoral injection of t-PAu / PIC and t-PAu / PIC combined with NIR irradiation in LLC mice was mainly studied by loading AuNC with vesicles, cRGD modification, and combination with ICB therapy. The specific experimental arrangement is shown in A of Figure 3 . To simulate the challenge of the irradiation range during actual PTT, the treatment was started when the average tumor volume was 100-120 mm 3 , and the maximum tumor volume of each group reached 150 mm 3 . At this time, the laser spot was slightly smaller than the tumor cross-section, and a small part of the edge laser could not be irradiated.

[0058] Monitoring of LLC tumor growth curves showed that in the single intratumoral injection of PAu+L (photothermal only) group, tumor volume decreased to a very low level in the first three days, but recurrence began around day five, with the tumor growth slope even higher than that of PBS. Although the average tumor volume of mice treated with PPIC only (immunization only) and AuNC+PPIC+L groups was significantly smaller than that of the PBS group, the treatment effect was limited; even immediate photothermal exposure after intratumoral injection failed to exert its photothermal effect. In stark contrast, treatment in the PAu / PIC+L and t-PAu / PIC+L groups significantly inhibited tumor progression (****p, Figure 3 (B) By the third day, the tumors had basically disappeared. These two combined treatments resulted in 83% of the mice in their respective groups having their tumors eliminated within 20 days. Figure 3 (C). During this period, the mice's body weight did not change significantly ( Figure 3 (D). These results also demonstrate that nanomedicines co-loaded with gold nanoparticles and TLR3 agonists can remain in tumors for a longer period, solving the problem of existing AuNCs' ineffective tumor retention and exhibiting significantly superior therapeutic effects. 83% of mice treated with PAu / PIC+L or t-PAu / PIC+L were completely cured and showed no recurrence within 120 days. Figure 3 (E).

[0059] The results of treating LLC mice with these two therapies in combination with CTLA-4 antibodies (Ab, IV, 1 mpk) showed that the tumor suppression survival rate of mice treated with PAu / PIC+L+Ab was no different from that of the antibody-free group, while the t-PAu / PIC+L+Ab group achieved a 100% cure rate and 120 days of recurrence-free treatment. It is well known that Treg cells are key immunosuppressive cells regulating anti-tumor immunity. The expansion of Treg cells in the tumor microenvironment (TME) can migrate from the peripheral circulation and then be further polarized into the pro-tumor phenotype via tumor-derived cytokines or exosomes. It has been reported that if treatment fails to eradicate tumor cells, it can lead to unfavorable proliferation of Treg cells, resulting in tumor recurrence and metastasis. This invention utilizes t-PAu / PIC+L-mediated local hyperthermia to effectively clear tumors, eliminate tumor-associated Tregs, and promote checkpoint blockade immunotherapy, thereby achieving a 100% cure rate in LLC mice.

[0060] Following a single injection of t-PAu / PIC supplemented with NIR and Ab, the immunomodulatory effects of t-PAu / PIC+L+Ab on LLC mice were investigated. Figure 4 (Middle A). 48 hours after the last injection, the tumors in groups 5-7 were significantly smaller than those in the monotherapy group ( Figure 4 (Chinese B). CD80 + DCs and NKG2D +NK cells (active receptors for killing tumor cells) are central APCs in an effective immune response. Compared with other groups, the proportions of both were significantly increased in groups 6 and 7 in the TME (DC: 20%, NK: 2.1%, *p). Figure 4 (C, D), they effectively bridge innate immunity and acquired anti-tumor immunity, thereby increasing CD8 in the TME in groups 2-4. + The proportion of T cells increased by 2.1-5.9 times, and CD25... + The proportion of CTL increased by 2.4-8.2 times, IFN-γ + The proportion of CTL increased by 3.0-6.0 times, CD4 + The proportion of T cells increased by 2-5.3 times. Figure 4 (In EG). IFN-γ can upregulate the expression of MHC-I molecules, thereby promoting CTL recognition of tumor cells.

[0061] Furthermore, groups 6 and 7 simultaneously showed a significant downregulation of the levels of immunosuppressive TME-related cells (Treg, M2M / M1M, and MDSC). Figure 4 It also significantly increases the CTL / Treg ratio by 14-80 times, which is beneficial to the formation of an anti-tumor immune environment. Figure 4 (J, ****p). t-PAu / PIC+L+Ab showed a 3.5-fold (**p) higher CTL / Treg ratio than the non-targeted PAu / PIC+L+Ab group. Notably, the tPAu / PIC+L+Ab group had higher activation of dendritic cells and MHC II in lymph nodes (LN). + DC cell content, NKG2D in peripheral blood (PB) + NK cell infiltration was also higher. Figure 4 (Middle MO). Furthermore, IL-6 and IFN-γ concentrations were also highest in PB. The overall order was: t-PAu / PIC+L+Ab > PAu / PIC+L > PAu / PIC+L > AuNC+PPIC+L > PAu +L > PAu / PIC > PBS ( Figure 4 (Middle MQ). In the spleen, CTL, CD25 + CTL, CD4 + The number of T cells and the CTL / Treg ratio both followed the same trend and order. Figure 4 Interestingly, the t-PAu / PIC+L and t-PAu / PIC+L+Ab groups significantly reduced the incidence of MDSCs that were substantially upregulated in the PTT monotherapy group. Figure 5The results above indicated that t-PAu / PIC+L+Ab combination therapy could effectively induce DC cell activation and T cell related immune response, effectively improve the immune suppressed microenvironment, promote systemic immunity, and have a strong anti-tumor effect.

[0062] To analyze the memory T cells in the cured mice, PBS, t-PAu / PIC+L and t-PAu / PIC+L+Ab groups were selected, and the LLC mice were treated according to the above described administration and light exposure schedule Figure 5 A). The tumor volume, body weight, state of the mice and the survival of the mice were monitored in the same way. On the 90th day, the cured mice were again inoculated with the same number of LLC cells, and the tumor growth was monitored (the newly inoculated LLC cells of the mice with PBS were used as the control). On the 60th day thereafter, the cured mice were sacrificed, and the abdominal blood was taken for blood biochemical and routine blood tests and analysis of memory T cells, and the spleen was taken, weighed and treated for detection of memory T cells therein. The specific operation was as follows: after the spleen was ground, the red blood cells were lysed with ACK, and the peripheral blood was directly lysed. The corresponding staining flow cytometry antibodies were added to the two kinds of single cell suspensions, and the flow cytometry was determined, and the FlowJo_V10 software was used to analyze the contents of central memory T cells (T EM , CD62L + CD44 + ) and effector memory T cells (T CM , CD62L - CD44 + ).

[0063] The tumor growth, body weight and survival curves of the LLC mice showed that the tumor growth of the three groups in this experiment was basically the same as above, and the tumors of the two treatment groups disappeared in the first 30 days Figure 5 B); the mice treated with t-PAu / PIC+L and t-PAu / PIC+L+Ab had a final cure rate of 75% and 100%, respectively, and the body weight of the mice was basically unchanged Figure 6 C, D). The results verified the reproducibility of the high therapeutic effect of the combination therapy strategy of the nano-drug.

[0064] On the 90th day of treatment, the two groups of cured mice were in good condition, and the same number of LLC cells were subcutaneously inoculated again, and the other C57 mice only received LLC cell inoculation as the control group (control). The results showed that the tumor of the control group of mice grew rapidly, while all the cured mice resisted the reattack of LLC cells by 100%, and no tumor was formed Figure 6(A). It can be observed that the t-PAu / PIC+L group showed slow tumor growth in the first 7 days, but the tumors were automatically cleared around day 7; while the tumors in the t-PAu / PIC+L+Ab group showed almost no growth. The results confirm that mice treated with t-PAu / PIC+L and t-PAu / PIC+L+Ab not only cleared tumors but also developed memory to resist the recurrence of tumors.

[0065] On day 60 (day 150) after revaccination, all mice were sacrificed, and peripheral blood and spleens were collected for flow cytometry analysis of memory T cells, primarily T cells. EM (Effective memory T cells) and T CM (Central memory T cells). The results showed that both treatments increased the number of memory T cells in mice compared to the PBS group. Figure 3 (C, D). Among them, CD4 in peripheral blood and spleen of the t-PAu / PIC+L+Ab group + T CM and CD8 + T CM The levels of CD4+ were significantly increased in peripheral blood and spleen of the t-PAu / PIC+L group; while the levels of CD4+ in peripheral blood and spleen of the t-PAu / PIC+L group were significantly increased. + T EM and CD8 + T EM The content of these cells is higher. Memory T cells are key to maintaining long-term immunity; they are mainly divided into T cells that migrate to tissues surrounding inflammation and exhibit immediate effector functions. EM T cells located in the T cell region of secondary lymphoid organs CM Two categories. T EM Upon encountering the antigen again, it can rapidly produce effector cytokines; while T CM They have almost no effector function, but possess a high proliferative capacity and can rapidly proliferate and differentiate into effector cells upon antigen stimulation. Both types of immune cells can remain in the body for years or even a lifetime.

[0066] In accordance with Figure 7 On day 120 of treatment with α1, some of the cured mice were subcutaneously inoculated again with LLC cells or GBM (glioblastoma) cells. As with previous experiments, all previously cured mice re-inoculated with LLC cells did not develop LLC tumors. Figure 7(A). Among mice re-inoculated with GBM cells, only the t-PAu / PIC+L+Ab or PAu / PIC+L+Ab groups, which had previously received combined CTLA-4 antibody treatment, were 100% resistant to tumor formation. Mice previously treated with PAu / PIC+L and t-PAu / PIC+L showed essentially no change in tumor size within 10 days after GBM cell inoculation, but the GBM tumors subsequently grew slowly, although at a much slower rate than the tumors in the PBS group. Figure 5 (B)

[0067] The above experimental results demonstrate that the nanomedicine of the present invention can generate strong immune memory after photothermal immunotherapy. This is an important ability of the immune system to remember pathogens in order to prevent subsequent disease infections. t-PAu / PIC showed even stronger and broader memory after being combined with CTLA-4 antibody.

[0068] Example 5: Systemic toxicity study of photoimmunotherapy in LLC mice

[0069] Next, the photothermal immunotherapy of nanomedicines was studied (by...). Figure 8 The treatment showed systemic toxicity in LLC mice. Results indicated that, compared to the healthy group, the treatment group exhibited a significantly increased proportion of neutrophils (Neut) (**p) and a significantly decreased monocyte count (Mono) (****p). Neutrophils are a type of white blood cell with phagocytic and bactericidal functions, while Mono counts decrease due to the immune response during disease onset. Mice in the PBS group showed significantly decreased alanine aminotransferase (ALT) and increased alanine aminotransferase (ALP), indicating liver damage. Furthermore, elevated creatinine (CREA) also suggested kidney impairment. Therefore, the changes in liver and kidney function indicators were caused by the tumor. The treatment group showed no significant side effects and, to some extent, alleviated liver and kidney damage and activated the immune system. After treatment, various indicators increasingly approached those of healthy mice. Figure 9 ).

[0070] Example 6: t-PAu / PIC for photothermal immunotherapy in mice bearing 4T1-luc triple-negative breast cancer

[0071] To establish a mouse subcutaneous 4T1-luc triple-negative breast cancer (TNBC) model, 4T1-luc cells (2 × 10⁻⁶) were used. 5 Each mouse was resuspended in PBS containing 30% matrigel, and 50 μL was injected subcutaneously above its right hind leg. Tumors could grow to 100-120 mm in approximately 8 days. 3 Left and right. To establish a mouse subcutaneous 4T1-luc distal tumor model to simulate metastatic tumors, the same number of 4T1-luc cells were subcutaneously inoculated above the left hind leg of the mouse on day 3 after the above inoculation.

[0072] To investigate the efficacy of t-PAu / PIC-based photothermal immunotherapy in 4T1-luc model, the mice were grouped and dosed when the primary tumors reached 100-120 mm 3 in volume. The first dosing day was recorded as day 0. The mice were divided into 5 groups (n = 6) and dosed i.t. starting on day 0. The mice in the photothermal group received NIR light (808 nm, 2 W / cm 2 , 5 min) once 30 min after i.t. dosing on day 0. The mice in the CTLA-4 antibody (Ab) group were dosed on days 1, 4, and 7 (1 mpk, i.v.). The groups were PBS, AuNC + PPIC + L, t-PAu + L, t-PAu / PIC + L, and t-PAu / PIC + L + Ab. The PIC dose was 1.25 mpk, and the Au dose was 0.5 mpk. The methods for measuring the tumor volume and body weight of the mice and the survival curve were the same as those for the LLC mouse treatment.

[0073] To investigate the ability of t-PAu / PIC-based photothermal immunotherapy to inhibit distant tumors (Distant Tumor), the mice were grouped and dosed when the primary tumors reached 100-120 mm 3 in volume. The groups were PBS, t-PAu / PIC + L, and t-PAu / PIC + L + Ab, and the experimental protocol was the same as above. The mice were only dosed i.t. and irradiated on the right primary tumor, and the distant small tumors were not treated. The tumor volume, body weight, and state of the mice were monitored every 3 days. The methods for determining death and volume were the same as above.

[0074] After the 4T1-luc mouse model was established subcutaneously in BALB / c mice for 8 days, the mice were dosed according to the A protocol in Figure 9 , with the same doses as for the LLC mouse treatment. The experimental results showed that the AuNC + t-PPIC + L treatment had limited tumor inhibition and survival extension for the 4T1-luc mice (B, C in Figure 9 ). The mice that received t-PAu + L targeted photothermal treatment showed strong tumor inhibition, but some tumors recurred rapidly, similar to the LLC mice. The mice in the t-PAu / PIC + L and t-PAu / PIC + L + Ab groups that received photothermal immunotherapy showed significantly enhanced tumor inhibition. One and two mice, respectively, had their tumors regress around days 5-7 (B, C in Figure 9 ). There were no significant changes in the body weight of the mice in each group during the treatment (D in Figure 9Figure 6. Survival curves of mice treated with PBS, AuNC + t-PPIC + L, t-PAu + L, t-PAu / PIC + L and t-PAu / PIC + L + Ab. The median survival time (MST) of mice treated with PBS, AuNC + t-PPIC + L, t-PAu + L was 18, 23.5 and 27 days, respectively. The MST of mice treated with t-PAu / PIC + L and t-PAu / PIC + L + Ab was 38.5 and 43 days, respectively. Figure 10 Figure 7. The therapeutic effect of t-PAu / PIC + L and t-PAu / PIC + L + Ab on the metastatic tumor. The metastatic tumor was inoculated on the opposite side of the primary tumor. The primary tumor was treated with t-PAu / PIC + L and t-PAu / PIC + L + Ab, respectively. The metastatic tumor was not treated.

[0075] Figure 8. The therapeutic effect of t-PAu / PIC + L and t-PAu / PIC + L + Ab on the metastatic tumor. The metastatic tumor was inoculated on the opposite side of the primary tumor. The primary tumor was treated with t-PAu / PIC + L and t-PAu / PIC + L + Ab, respectively. The metastatic tumor was not treated. Figure 10 Figure 9. The therapeutic effect of t-PAu / PIC + L and t-PAu / PIC + L + Ab on the metastatic tumor. The metastatic tumor was inoculated on the opposite side of the primary tumor. The primary tumor was treated with t-PAu / PIC + L and t-PAu / PIC + L + Ab, respectively. The metastatic tumor was not treated. 3 Figure 10. The therapeutic effect of t-PAu / PIC + L and t-PAu / PIC + L + Ab on the metastatic tumor. The metastatic tumor was inoculated on the opposite side of the primary tumor. The primary tumor was treated with t-PAu / PIC + L and t-PAu / PIC + L + Ab, respectively. The metastatic tumor was not treated. 3 Figure 11. The therapeutic effect of t-PAu / PIC + L and t-PAu / PIC + L + Ab on the metastatic tumor. The metastatic tumor was inoculated on the opposite side of the primary tumor. The primary tumor was treated with t-PAu / PIC + L and t-PAu / PIC + L + Ab, respectively. The metastatic tumor was not treated. Figure 10 Figure 12. The therapeutic effect of t-PAu / PIC + L and t-PAu / PIC + L + Ab on the metastatic tumor. The metastatic tumor was inoculated on the opposite side of the primary tumor. The primary tumor was treated with t-PAu / PIC + L and t-PAu / PIC + L + Ab, respectively. The metastatic tumor was not treated. Figure 11 Figure 13. The therapeutic effect of t-PAu / PIC + L and t-PAu / PIC + L + Ab on the metastatic tumor. The metastatic tumor was inoculated on the opposite side of the primary tumor. The primary tumor was treated with t-PAu / PIC + L and t-PAu / PIC + L + Ab, respectively. The metastatic tumor was not treated.

[0076] Figure 14. The therapeutic effect of t-PAu / PIC + L and t-PAu / PIC + L + Ab on the metastatic tumor. The metastatic tumor was inoculated on the opposite side of the primary tumor. The primary tumor was treated with t-PAu / PIC + L and t-PAu / PIC + L + Ab, respectively. The metastatic tumor was not treated. ​ Figure 15. The therapeutic effect of t-PAu / PIC + L and t-PAu / PIC + L + Ab on the metastatic tumor. The metastatic tumor was inoculated on the opposite side of the primary tumor. The primary tumor was treated with t-PAu / PIC + L and t-PAu / PIC + L + Ab, respectively. The metastatic tumor was not treated.

[0077] The present application proves that the cRGD-mediated vesicle nano-drug co-loaded with gold nanoclusters and TLR3 agonist (t-PAu / PIC) can induce safe and efficient immune response under NIR light, and only one intratumoral injection and NIR irradiation can strongly eliminate the tumor of LLC mice slightly larger than the laser spot. The t-PAu / PIC+L+Ab combined with CTLA-4 antibody can further significantly prolong the survival time of mice, and the cure rate of mice reaches 100%, and the combination therapy can significantly increase the infiltration of mDC cells, CTL and NK cells and can down-regulate the proportion of immunosuppressive MDSCs, M2M and Treg. The cured mice can resist tumor growth by 100% after being attacked by LLC and GBM tumor cells again, showing long-term, effective and relatively broad-spectrum memory effect. Immunological analysis shows that t-PAu / PIC+L or t-PAu / PIC+L+Ab treatment can significantly increase the content of memory T cells in vivo. In addition, t-PAu / PIC+L+Ab treatment of TNBC model 4T1-luc mice can also cure 33% of the mice and significantly prolong the life of the mice. The treatment strategy based on t-PAu / PIC+L can solve the shortcomings of traditional PTT, including tumor recurrence and metastasis, possible damage to healthy tissues and inability to work outside the laser irradiation area. The integrin-targeted nano-drug t-PAu / PIC designed in the present application provides a potential strategy for curing lung cancer for efficient photothermal immunotherapy of tumors and combined treatment with ICB.

Claims

1. A nano-drug co-loading photothermal therapeutic agent and TLR agonist, comprising a reduction-sensitive disulfide cross-linked vesicle and the TLR agonist and photothermal therapeutic agent loaded therein, characterized in that, The reduction-sensitive disulfide cross-linked vesicle is a non-targeted reduction-sensitive disulfide cross-linked vesicle or a targeted reduction-sensitive disulfide cross-linked vesicle; the TLR agonist is poly (I:C); the photothermal therapeutic agent comprises a gold nanoparticle material; the amphiphilic block polymer, the TLR agonist and the photothermal therapeutic agent are mixed in a solution to obtain the nano-drug co-loaded with the photothermal therapeutic agent and the TLR agonist; the amphiphilic block polymer is a hydrophilic segment-P (B-DTC)-cationic segment, or the amphiphilic block polymer is a hydrophilic segment-P (B-DTC)-cationic segment and A-hydrophilic segment-P (B-DTC); wherein A is a targeting molecule, B is a cyclic ester or a cyclic carbonate monomer unit; when the amphiphilic block polymer is a hydrophilic segment-P (B-DTC)-cationic segment and A-hydrophilic segment-P (B-DTC), the molar percentage of A-hydrophilic segment-P (B-DTC) in the polymer is 0-40%, excluding 0; the mass ratio of the TLR agonist to the photothermal therapeutic agent is 1: (0.1-50); the hydrophilic segment is PEG, the cationic segment is one of spermine and small molecular weight PEI, and DTC is dithiopentane tris-methylene carbonate.

2. The method of claim 1, wherein the method of preparing the nano medicine co- loaded with the photothermal therapeutic agent and the TLR agonist is characterized by, The method comprises the following steps: mixing the amphiphilic block polymer, the TLR agonist and the photothermal therapeutic agent in a solution to obtain the nano-drug co-loaded with the photothermal therapeutic agent and the TLR agonist.

3. A combination drug, characterized by comprising: The method comprises the following steps: mixing the amphiphilic block polymer, the TLR agonist and the photothermal therapeutic agent in a solution to obtain the nano-drug co-loaded with the photothermal therapeutic agent and the TLR agonist.

4. Use of the nano-drug co-loaded with the photothermal therapeutic agent and the TLR agonist according to claim 1 or the combination drug according to claim 3 in the preparation of a drug for resisting lung cancer.

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