Loading APD1 Targeted Phase-shifted Nanoparticles For Photothermal Treatment And Synergistic Immunotherapy Of Malignant Melanoma

PART ? PREPARATION AND CHARACTERIZATION OF LOADING aPD1 TARGETED PHASE-SHIFTED NANOPARTICLESObjectives To prepare targeted phase-shifted nannoparticles loading aPD1,PFP and iron oxide(GOP@ aPD1 NPs),characterize their basic properties,photothermal ability,phase transitional characteristics,drug loading and drug release effect.Methods Firstly,the adhesive polypeptide(Gly-Arg-Gly-Asp-Ser,GRGDS)-targeted polylactic acid glycolic acid/polyethylene glycol polymer material(PLGA-PEG-GRGDS)was prepared and its chemical structure was identified.GOP@aPD1 NPs were prepared by improved three-step emulsification and rotary evaporation methods.The basic physical and chemical properties of the nanoparticle,including particle size,zeta potential,morphological structure and absorption spectrum were detected.The particle size changes of nanoparticles in PBS and fetal bovine serum containing PBS were measured at different time points after preparation(0.5 h,1 d,3 d,7 d).Flow cytometry was used to detect the attachment of nanoparticles to GRGDS antibodies.The phase transition of nanoparticles at different temperatures(room temperature,37 ? and 45 ?)was observed under light microscope.The photothermal characteristics of the nanoparticles were measured under different concentrations of 660 nm laser irradiation.Ultrasound-enhanced enhanced signal at different time points(2 min,6 min,10 min,12 min)during 660 nm laser irradiation were recorded.The aPD1 drug content in the GOP@aPD NPs and the release of aPD1 drug by photothermal effect were detected by enzyme-linked immunosorbent assay(ELISA).GOP@aPD nanoparticles loading and releasing aPD1 were also observed by laser confocal.Results The structure of the polymer PLGA-PEG-GRGDS was confirmed by hydrogen spectroscopy.The GOP@aPD1 nanoemulsion was brown.Scanning electron microscopy(SEM)showed the nanoparticles have a spherical structure with uniform size and good dispersion.Transmission electron microscope(TEM)showed that the spotted Fe3O4 particles were uniformly distributed in the nanoshell.Malvern particle size analyzer measured GOP@aPD1 NPs with an average particle size of 218 nm and an average potential of-5 mV.After placing in PBS and 10% fetal bovine serum PBS solution at 4 ? for 7 days,the particle size of GOP@aPD1 NPs did not change significantly,which proved the stability of the nanoparticles.GOP@aPD1 NPs connected with more GRGDS antibodies compared to non-targeted nanoparticles measured by flow cytometry,demonstrating that the surface of the nanoparticles were linked to GRGDS polypeptide.The encapsulation efficiency of Fe3O4 in GOP@aPD1 NPs was 63% by atomic absorption spectrometry.It was observed by light microscopy that the nanoparticles remained almost stable at room temperature and 37 ?.But at 45 ?,the nanoparticles underwent gas phase change,the volume increased,and mutual fusion and rupture.The phase transition temperature of GOP@aPD1 NPs is about 45 ?.The photoacoustic absorption spectrum showed that GOP@aPD1 NPs has a strong absorption peak around 700 nm.The GOP@aPD1 NPs were diluted to 1.25-10 mg/mL according to the PLGA-PEG-GRGDS concentration.Under the irradiation of 660 nm laser,the warming effect of different concentrations of GOP@aPD1 NPs was obvious,and the elevated temperature was positively correlated with the concentration of nanoparticles.The 5 mg/mL group could be heated up to about 45 ?.Under the continuous irradiation of 660 nm laser,the ultrasonic enhanced signal of the nanoparticle gradually rised and decreased with time,and the ultrasonic enhanced signal of the nanoparticle was the largest when irradiated for 10 min,indicating that liquid-gas phase change peaks with the laser irradiation time for 10 min.The drug loading of aPD1 drug in GOP@aPD1 NPs was 1.56%(w/w)and the entrapment efficiency was 98.9 % by ELISA.The in vitro drug release test showed that the aPD1 drug in GOP@aPD1 NPs was released rapidly within 2 h under laser irradiation,and the drug release was flat after 6 h.Laser confocal microscopy showed that the red fluorescence of the DiI-labeled GOP@aPD1 NPs and the green fluorescence of the FITC-labeled aPD1 co-localized a yellow fluorescent signal before laser irradiation,while the red fluorescence and the green fluorescence were separated after laser irradiation.It is indicated that the photothermal effect produced by laser irradiation promotes the release of aPD1 drug from GOP@aPD1 NPs.Conclusion In this experiment,GOP@aPD1 NPs were prepared by emulsification method.The morphology was regular,the size was relatively uniform,the properties were stable,and the aPD1 and Fe3O4 were loaded.GOP@aPD1 NPs had good photothermal capability and liquid-gas phase change ability,and could release the aPD1 after be irradiated by 660 nm laser.PART 2 TARGETING ABILITY TO MELANOMA AND DISTRIBUTION IN VIVO of LOADING aPD1 TARGETED PHASE-SHIFTED NANOPARTICLES Objectives To observe the targeting ability of melanoma cells B16F10 by GOP@aPD1 NPs in vitro and in vivo,as well as the biodistribution of the nanoparticles,circulation time in the blood and biosafety in vivo.Methods GOP@aPD1 NPs and non-targeted NPs were prepared fistly.In vitro,laser confocal microscopy was used to observe the colocalization of Di I-labeled nanoparticles and DAPI-stained B16F10 cells.Flow cytometry was used to measure the connectivity of Di I-labeled nanoparticles and B16F10 cells after co-incubation different time.CCK8 method was used to observe the changes of cell viability of B16F10 cells after incubation of different concentrations(2.5 mg/ml,5 mg/ml,10 mg/ml,15 mg/ml)of GOP@aPD1 NPs.In vivo,the C57B6 mouse B16F10-luc melanoma model was first established.When the tumor volume was about 100 mm3,Di R-labeled GOP@aPD1 NPs and non-targeted NPs were injected into the tail vein of C57B6 mice.Fluorescence images were acquired in a Xenogen IVIS bioluminescence imaging system,and the signal intensity of tumors and vital organs were analyze.C57B6 mice were injected with different concentrations(5 mg/ml,10 mg/ml)of GOP@aPD1 NPs.After 14 days the serum of CK,LDH-L,AST,ALT,BUN and CR were analyzed.The biosafey of GOP@aPD1 NPs was observed.GOP@aPD1 NPs were prepared using PerCP/Cy5.5 labeled aPD1.PerCP/Cy5.5-labeled GOP@aPD1 NPs and PerCP/Cy5.5-labeled free aPD1 were injected into the tail vein of tumor-bearing C57B6,respectively.Tumors and vital organs were taken at different time points(6 h,10 h)after injection.Frozen sections were prepared.The red fluorescence signal of PerCP/Cy 5.5 in each tissue section was observed under laser confocal microscopy.The signal intensity was analyzed by Image-Pro Plus 6.0 software.Mouse blood was collected at different time points(0.5 h,1 h,2 h,4 h,6 h,8 h,12 h,24 h)after injection of PerCP/Cy5.5-labeled GOP@aPD1 NPs,and fluorescence spectrophotometer analysis was performed.Results In the first part,the red fluorescence of the Di I-labeled GOP@aPD1 NPs localized around the B16F10 cells was more than the non-targeted NPs observed by laser confocal microscopy.After incubation with B16F10 cells 2 h and 10 h,GOP@aPD1 NPs connecting with cells were significantly higher than non-targeted NPs(p < 0.01)by flow cytometry.There was no significant change in cell viability after incubation with B16F10 cells at different concentrations of GOP@aPD1 NPs(2.5 mg/ml,5 mg/ml,10 mg/ml,15 mg/ml),but the cell viability was significantly reduced after laser irradiation,decreased as the concentration of GOP@aPD1 NPs increased.In vivo targeting experiments,GOP@aPD1 NPs gradually enriched in tumor sites through high permeability and retention(EPR)effects and active targeting,and fluorescence signals of tumor sites were observed in a Xenogen IVIS bioluminescence imaging system,which were gradually increased with the passage of time,reached the highest peak after 6 hours of administration,and weakened after 24 hours.In the non-targeted NPs group,the fluorescence signals of the tumor site at each time point were significantly lower than that of the GOP@aPD1 NPs group.There were no significant differences in serum CK,LDH-L,AST,ALT,BUN and CR after 14 days I.V.of different concentrations of GOP@aPD1 NPs(5 mg/ml,10 mg/ml).In the second part of the experiment,tumor-bearing mice were respectively injected of PerCP/Cy5.5-labeled GOP@aPD1 NPs and PerCP/Cy5.5-labeled free aPD1.At 6 h and 24 h post injection,the fluorescent signal GOP@aPD1 NPs in the tumor tissues were observed by laser confocal microscopy.However,in the PerCP/Cy5.5-labeled free aPD1 group,red fluorescent models in tumor tissues were hardly observed by laser confocal microscopy.The fluorescence signal intensity analysis in tumor tissues showed that the fluorescence intensity of aPD1 in the tumor tissues of GOP@aPD1 NPs group was significantly higher than that of the free aPD1 group both at 6 h and 24 h.When 24 h after I.V.injection of PerCP/Cy5.5-labeled GOP@aPD1 NPs in the tumor-bearing mice,fluorescence signals in the blood was still detected by fluorescence spectrophotometer.The half-life of GOP@aPD1 NPs circulating in the body is about 4.39 h.Conclusion The prepared GOP@aPD1 NPs could be enriched in tumor tissues through EPR effect and active targeting,and achieved targeted melanoma delivery of aPD1 drug,with long in vivo circulation time and good biosafety,laying the foundation for the next in vivo therapeutic experiment.PART 3 PHOTOTHERMAL-MEDIATED LOADING aPD1 TARGETED PHASE-SHIFTED NANOPARTICLES ON THE TREATMENT OF MELANOMA AND THE MECHANISIOM OF SYNERGISTIC IMMUNOTHERAPY Objectives To study the effect of photothermal-mediated GOP@aPD1 NPs therapy on the treatment of melanoma and its related mechanism of synergistic aPD1 immunotherapy.Methods To observe photo-thermal effect in vivo after photothermal-mediated GOP@aPD1 NPs therapy,the C57B6 mice B16F10-luc melanoma model were established and different treatments were initiated when the tumor volume was approximately 80 mm3.Tumor-bearing mice were randomly divided into 3 groups,control group(no treatment),PTT group and GOP@aPD1+PTT group.The GOP@aPD1+PTT group was injected with GOP@aPD1 NPs(~200 ?L,5 mg/m L).After 6 hours,the tumors were irradiated with 660 nm laser(0.1 W/cm2,10 min).The PTT group was given only 660 nm laser irradiation(0.1 W/cm2,10 min)on tumor tissue.The temperature of the tumor site was recorded using an infrared imager.Serum was taken at different time points(24 h,72 h,168 h)after treatment,and the levels of inflammatory factors IL-6,TNF-? and IFN-? were analyzed by ELISA.To observe the effect of photothermal-mediated GOP@aPD1 NPs therapy on melanoma in vivo,GOP@aPD1 NPs,single-loaded Fe3O4 NPs and single-loaded aPD1 NPs were first prepared.Among them,the single-loaded Fe3O4 NPs did not contain aPD1,and the single-loaded aPD1 NPs ddi not contain Fe3O4.The C57B6 mice B16F10-luc melanoma model were established and began to be treated differently when the tumor volume was approximately 80 mm3.The mice were randomly divided into 8 groups,1.control group(saline),2.free aPD1 group,3.PTT group,4.free aPD1 + PTT group,5.single-loaded Fe3O4 NPs + PTT group,6.single-loaded aPD1 NPs +PTT group,7.GOP@aPD1 group and 8.GOP@aPD1+PTT group.Group 2 and group 7 were only injected with free aPD1 and GOP@aPD1 NPs respectively;group 3 were given only 660 nm laser irradiation(0.1 W/cm2,10 min);group 4,group 5,group 6 and group 8 were injected with free aPD1,single-loaded Fe3O4 NPs,single-loaded aPD1 NPs and GOP@aPD1 NPs,then the tumors were irradiated with 660 nm laser(0.1 W/cm2,10 min)after 6 hours.After different treatments,the tumor volume,body weight and survival time of mice were observed.Mice were injected intraperitoneally with luc fluorescein substrate at different time points(0 d,7 d,14 d)after treatment,and tumor fluorescence signals were collected in Xenogen IVIS bioluminescence imaging system.After 7 days of treatment,mice were randomly sacrificed in each group.Heart,liver,spleen,lung,kidney and tumor tissues were collected for hematoxylin-eosin(HE)staining,and TUNEL immunofluorescence staining was performed on tumor tissues to detect proliferation and apoptosis of tumor cells.To investigate the mechanism by which photothermal-mediated GOP@aPD1 NPs treatment potentiate aPD1 immunotherapy.In the third part of the experiment,the mice were grouped and processed in the same way as the second part.The mice were sacrificed after 7 days of treatment,and the tumor tissues were collected for flow cytometry and immunofluorescence staining to detect the number of tumor infiltrating T lymphocytes,CD8+ T lymphocytes and CD4+ T lymphocyte subsets.Results The results of photothermal reaction in vivo showed that the maximum temperature of the tumor site reached about 42 ? and 45 ? in the PTT group and the GOP@aPD1+PTT group respectively after laser irradiation,and both of two groups produced photothermal effects.However,ELISA analysis of serum inflammatory factors IL-6,TNF-? and IFN-? showed that the levels of serum inflammatory factors in the GOP@aPD1+PTT group were significantly higher than those in the PTT group.At 72 h after treatment,the serum IFN-? level in the GOP@aPD1+PTT group was 3.5 times higher than in the PTT group.At 168 h after treatment,the serum IFN-? level in the GOP@aPD1+PTT group was 3 times higher than in the PTT group.These results demonstrated that photothermal-mediated GOP@aPD1 NPs treatment could produce tumor photothermal effect and effectively activated the level of inflammatory response in vivo.In the treatment of malignant melanoma in vivo,tumor growth in the free aPD1 group,PTT group,single-loaded Fe3O4 NPs+PTT group and GOP@aPD1 group were not significantly inhibited,and there was no significant difference in tumor growth with the control group.Tumor growth was inhibited partly in the free aPD1+PTT group and single-loaded aPD1 NPs +PTT group.However,tumor growth was completely inhibited in the GOP@aPD1+PTT group.In vivo fluorescence experiments showed that the tumor fluorescence intensity was the weakest in the GOP@aPD1+PTT group at 14 d after treatment.Animals in the GOP@aPD1+PTT group survived for more than 35 days,while the average survival time of the animals in the free aPD1+PTT group and single-loaded aPD1 NPs+PTT group was 19 days and 20 days,respectively.HE staining of tumor tissue showed that a large number of tumor cells were necrotic in the tumor tissue of GOP@aPD1+PTT group,no intact cell structure,red stained no structural area under the microscope,and TUNEL immunofluorescence staining showed that the apoptosis fluorescence of the tumor tissue was the highest.There was no obvious abnormality in HE staining of the main organs of each group,which proved the biosafety of the treatment.Laser confocal microscopy showed that there was almost no tumor infiltrating T lymph infiltration in the tumor tissue sections of the control group,while a large number of CD3+ T lymphocytes,CD8+ T lymphocytes and CD4+ were observed in the tumor tissue sections of the GOP@aPD1+PTT group.The results of low cytometry analysing the number and subpopulation of infiltrating T lymphocytes in the tumor microenvironment showed that the number of tumor infiltrating CD3+ T lymphocytes in the GOP@aPD1+PTT group was the highest among each groups,and the proportion of CD8+ T lymphocytes was the highest among each groups.It was proved that photothermal-mediated GOP@aPD1 NPs treatment effectively increased the amount of anti-tumor T cells in the tumor microenvironment,thereby enhancing the efficacy of aPD1 immunotherapy.Conclusion Photothermal-mediated GOP@aPD1 NPs treatment improved the level of anti-tumor inflammatory response in vivo,increaseed the amount of T lymphocytes in tumor microenvironment,and achieved the transition from immune "cold" tumors to immune "hot" tumors,thereby enhancing immunotherapy of aPD1.The treatment effectively inhibited the growth of melanoma and provided a new way for melanin treatment.