Experimental Study Of Targeted Molecular Probes-based Tumor Immunosuppression Alleviating For Tumor Photothermal Synergistic Immunotherapy

PART Ⅰ SYNTHESIS,PHYSIO-CHEMICAL PROPERTIES AND BIOSAFETY EVALUATION OF Nano-IR-SB@LipObjective To prepare a IR780 and SB-505124(an inhibitor of transforming growth factor-β)loaded photothermal-immunomodulatory liposome(designed as Nano-IR-SB@Lip).Test the physio-chemical properties,UV-vis spectral properties,in vitro photothermal conversion potential and verify their biosafety in vivo.Methods Nano-IR-SB@Lip was prepared by the filming-rehydration method,and the characterizations including average diameter,surface potential,morphology were determined.The UV spectrophotometer was employed to measure the absorbance values of IR780 and SB-505124 at different concentrations.Then the encapsulation efficacies of IR780 and SB-505124 were analyzed by the standard curves.With laser irradiation,the temperature change of Nano-IR-SB@Lip was investigated by thermal infrared imager.Meanwhile,the Balb/c mice were intravenously injected with NanoIR-SB@Lip(200 μL,4 mg/m L).After 1 d,7 d,14 d,21 d,28 d of injection,the blood of mice were collected to analysis the change of biochemical index and blood routine, and dissected the major organs(heart,liver,spleen,lung,kidney)for hematoxylin-eosin staining(H&E)staining to assess the biosafety of Nano-IR-SB@Lip.Results The obtained Nano-IR-SB@Lip presented uniform spherical morphology under transmission electron microscope(TEM).The Nano-IR-SB@Lip were well dispersed in aqueous solution,without obvious agglomeration.The average diameter of Nanno-IR-SB@Lip was 263.10 ± 59.22 nm detected by dynamic light scattering.And the surface Zeta potential was-17.20 ± 5.99 m V.It was found that the IR780 and SB-505124 presented absorption peaks at 780 nm and 320 nm respectively,with obvious concentration dependence.In addition,the encapsulation efficacies of IR780 and SB-505124 were 80.49 ± 2.30% and 53.09 ± 9.93% respectively,which were measured by the corresponding standard-curves.After laser irradiation,the temperature of Nano-IR-SB@Lip suspension increased with irradiation time.Especially,after Nano-IR-SB@Lip injection,the blood routine indexes,liver function,kidney function of mice showed negligible change during observation period.The results of H&E staining showed that the cell morphology of main organs were normal.Conclusion Nano-IR-SB@Lip were successfully fabricated,with uniform size,regular morphology and excellent photothermal conversion properties.And Nano-IRSB@Lip possess highly biosafety,presenting negligible toxic and side effects on mice.PART Ⅱ IN VITRO PHOTOTHERMAL THERAPY AND IMMUNE STIMULATING ACTIVITY OF Nano-IRSB@LIPObjective To investigate the phagocytosis,deep penetration,and mitochondrion targeting prosperity of Nano-R-SB@Lip.To study therapeutic efficacy of Nano-IRSB@Lip-mediated photothermal therapy(PTT)against 4T1 tumor cells and explore the tumor associated antigens(TAAs)releasing ability stimulated by Nano-IRSB@Lip-based PTT.Methods The phagocytosis of Nano-IR-SB@Lip were qualitatively and quantitatively by laser confocal microscope(CLSM)and flow cytometry.Then 3D tumor sphere models were established to investigate the distribution of Nano-IRSB@Lip in tumor spheres after co-incubated with liposomes.After incubation with Nano-IR-SB@Lip,4T1 cells were stained by a mitochondrial fluorescent probe to observe the co-localization of liposomes and mitochondria under CLSM.The cellkilling effect of Nano-IR-SB@Lip combined with laser irradiation was analyzed by a CCK-8 kit and Calcein acetoxymethyl ester(Calcein-AM)/propidium iodide(PI)costaining.After treated with PTT,the expression of calreticulin(CRT),high mobility group box protein 1(HMGB1)and ATP of 4T1 cells were assessed.Results After incubation with Nano-IR-SB@Lip for 4 hours,a large amount of Nanno-IR-SB@Lip were phagocytized by cells under CLSM.Flow cytometry analysis results showed that the fluorescence intensity was enhanced with incubation time of Nano-IR-SB@Lip.In the 3D tumor sphere model,Nano-IR-SB@Lip were distributed on the surface and inner of the tumor sphere.In addition,a lot of Nano-IR-SB@Lip aggregated in mitochondrial after stained with mitochondrial tracer,while the NanoSB@Lip(without IR780 loading)had no obvious aggregation in the mitochondrial region.Besides,the cell viability was lower than 10% when the cells received NanoIR-SB@Lip-based PTT and the cells presents obvious red fluorescence after live-dead co-staining,indicating cell death.Meanwhile,cells expressed amounts of CRT,HMGB1 under CLSM.Compared with the blank control group,the ATP release content of cells increased significantly after PTT assessed by ATP kit(p<0.001).Conclusion After co-incubation with 4T1 cells,Nanno-IR-SB@Lip showed excellent phagocytic capacity,deep penetration and mitochondrial targeting ability.The PTT-treated cells exhibiting strong killing efficacy,followed by a large number of TAAs releasing.PART Ⅲ STUDY ON PHOTOACOUSTIC/FLUORESCENCE DUAL-MODEL IMAGING OF Nano-IR-SB@LipObjective To study photoacoustic(PA)imaging performance of Nano-IRSB@Lip in vitro and to evaluate PA/fluorescence(FL)enhancement of Nano-IRSB@Lip on 4T1 tumor-bearing mice.Methods The Nano-IR-SB@Lip suspension was scanned by PA laser with excitation wavelength from 680 nm to 970 nm to obtain continuous PA imaging and detect the optimum wavelength.The PA images of Nano-IR-SB@Lip at different concentrations(0.4 mg/m L-2.0 mg/m L)were acquired and were quantitively analyzed.Then 4T1 tumor-bearing mice were established for in vivo PA imaging by intravenously injection Nano-IR-SB@Lip(200 μL,4 mg/m L).The PA images were recorded at different time points(pre-injection,1,2,4,6,24 and 48 h)and the corresponding PA signal intensities were measured.The in vivo FL imaging was performed in same manner by a small animal FL imaging system and evaluate the relationship of FL intensity with blood circulation time of Nano-IR-SB@Lip.Besides,the tumor tissues and major organs were dissected for ex vivo FL imaging,and the distribution differences in tumor and organs were analyzed.Results Nano-IR-SB@Lip showed maximum PA value at 805 nm,which was regarded as optimum excitation wavelength for following PA imaging.The in vitro PA imaging results showed that PA intensity increased with elevated Nano-IR-SB@Lip concentration,y=0.2841 x + 0.5789.In addition,the PA intensity in tumor region strengthened over time and reached to peak at 24 h with 2.7-fold signal enhancement.The in vivo FL followed the same tendency that the FL intensity reached to peak at 24h post-injection and then decreased at 48 h.Meanwhile,in the ex vivo imaging,it was found that the FL signal was higher than that in organs(p<0.05).Conclusion Nano-IR-SB@Lip can effectively accumulated in tumor tissues and enhance PA/FL imaging,being a potential dual-mode contrast agent.PART Ⅳ IN VIVO PHOTOTHERMALIMMUNOTHERAPY ASSISTED BY Nano-IR-SB@LipObjective To explore the in vivo immunomodulatory effect of Nano-IRSB@Lip on immunosuppressive microenvironment including CD8+T cell,regulatory T(Treg)cells and to evaluate the anti-tumor efficacy of Nano-IR-SB@Lip-based photothermal-immunotherapy combined with immune checkpoint inhibitor.Methods 4T1 tumor-bearing mice were established and received Nano-IRSB@Lip injection.The temperature change in tumor site was recorded during laser irradiation.Then the mice were randomly divided into following five groups: Control,Free SB,Nano-IR-SB@Lip,Nano-IR@Lip + Laser,Nano-IR-SB@Lip + Laser.At 7 days after above treatments,the ratio of CD8+ T cells and Treg cells in tumors were analyzed by flow cytometry and the distribution of these cells in tumor tissues were detected by immunofluorescence stanning.The serum of mice was collected at 1 day and 7 days to determine the secretion level of cytokines including TNF-α,IFN-γ and GZMS-B by ELISA assay.Next,to explore the anti-tumor efficiency of Nano-IR-SB@Lip,4T1 tumorbearing mice were assigned into six groups(the five groups are same as above mentioned except that add the Anti-CD4/CD8 + Nano-IR-SB@Lip + Laser group).All formulations(200 μL)were intravenously injected(the corresponding concentration of free SB was 0.1 mg/m L,and the concentration of Nano-IR@Lip or Nano-IR-SB@Lip were 4 mg/m L),followed by laser irradiation 1 day after administration.With the intervals of 2 days,the injections were repeated again.The tumor volumes and body weight of mice were monitored.The growth curves of tumor were drawn to observe the anti-tumor effect.The inhibition efficacies of each treatment were further assessed by pathological examination(H&E,TUNEL,PCNA stanning).Based on the above photothermal-immunotherapy,anti-PD-1 was further applied to improve the immunosuppressive microenvironment and observe the therapeutic effect of combined therapy.The anti-PD-1 was injected intraperitoneally 24 hours after laser irradiation,and the treatment was repeated every two days at a dose of 50 μg/mouse.During the treatment,the tumor growth and the survival of mice were recorded.Besides,the number of lung metastatic nodules in each group were detected.The inhibitory effect of combined treatments on tumor growth was systemically evaluated.Meanwhile,unilateral tumor models were used to evaluate the effect of combined therapy(Nano-IR-SB@Lip + Laser + Anti-PD-1)on anti-metastasis.The therapeutic efficacy was investigated by tumor growth curves and pathological analysis(H&E and PCNA staining).Results Under laser irradiation,the temperature in tumor site were rapidly increased after Nano-IR-SB@Lip injection.Compared to other groups,the ratio of CD8+ T cells in Nano-IR-SB@Lip + Laser group was the highest,while the ratio of Treg cells was the lowest.Meanwhile,Nano-IR-SB@Lip + Laser group presented strengthened signals of CD8+ T cells and weakened Treg cell signals.The levels of cytokine(TNF-α、IFN-γ、GZMS-B)in Nano-IR-SB@Lip + Laser group at 7 days was significantly higher than that at 1 day(p<0.05).Nano-IR-SB@Lip + Laser group showed the slowest growth rate and the best tumor inhibition effect(inhibition rate: 93.29 ± 6.96%).However,the antitumor effect was weakened(73.68 ± 15.24%)when the CD4+ and CD8+ T cells were inhibited.During the treatment,no obvious weight fluctuation occurred.The H&E staining showed that the structure of tumor cells was seriously destroyed after Nano-IRSB@Lip + Laser treatment.In the section of TUNEL staining,obvious apoptosis green fluorescence can be detected in Nano-IR-SB@Lip + Laser group.Otherwise,the red fluorescence signal in PCNA immunofluorescence section was weak,indicating the proliferation index was low.Stimulated by laser irradiation,Nano-IR-SB@Lip showed strongest inhibitory effect on tumor growth,which was further enhanced in the assisted by anti-PD-1,with a survival of 60 days.It was found that the number of pulmonary metastases in group of Nano-IR@Lip + Laser + anti-PD-1 was 2.80 ± 3.11,lower than that in control group(37.80 ± 8.81)and Nano-IR@Lip + Laser + PD-1 group(6.2 ± 0.84).In the mimic "metastatic tumor" model,it was observed that compared with the blank control group,Nano-IR@Lip + Laser + anti-PD-1 presented the best antitumor effect on “distant tumor”.The significant destruction of “primary” tumor cells was detected in H&E section after Nano-IR-SB@Lip + Laser + Anti-PD-1treatment,while anti-PD-1 or Nano-IR-SB@Lip + Laser treated-tumor cells remained relatively intact but partially cell damage.In the PCNA images,negligible red fluorescence was observed in NanoIR-SB@Lip + Laser + Anti-PD-1 group,suggesting low expression of PCNA.However,in the anti-PD-1 and Nano-IR-SB@Lip + Laser groups showed strong red fluorescence and high tumor cell proliferation rate.Conclusion Nano-IR-SB@Lip combined with laser irradiation can activate immune responses,regulate the immunosuppressive microenvironment,and realize tumor photothermal-immune antitumor therapy.Combined with anti-PD-1,it can both inhibit the growth of primary and distant tumors.