Experimental Study On Mutilmodal Imaging And Photothermal Therapy Of IR780-Loaded Target Multifunctional Nanoparticles

PART 1 PREPARATION AND CHARACTERIZATIONS OF IR780 LOADED MULTIFUNCTIONAL NANOPARTICLESObjective To prepare IR780-loaded multifunctional nanoparticles(cRGD-PLGA-IR780-PFP)and investigate their general properities including mean sizes,potentials,target effects and biosecurities.Methods PLGA-IR780-PFP nanoparticles were synthesized by a double-emulsion method and the targeted polypeptide cRGD was connected to the surface of PLGA-IR780-PFP nanoparticles by carbodiimide method,which finally formed the targeted multifunctional nanoparticles(cRGDPLGA-IR780-PFP).The general properties including mean sizes,potentials and structures were tested.The targeting ability of cRGD was systemically detected.In vitro cell uptake of nanoparticles was observed by confocal microscopy.The standard curve of IR780 was drawn and the encapsulation rate of IR780 in cRGD-PLGA-IR780-PFP was calculated.CCK-8 assay was used to detect the cytotoxicity of different concentrations of cRGD-PLGAIR780-PFP,cRGD-PLGA-IR780 and PLGA-IR780-PFP(the amount of IR780 in the three nanoparticles was the same)incubated with B16 cells for 24 h.BALB/c mice were randomly divided into 4 groups,including control group,1,7 and 14 days after injection of cRGD-PLGA-IR780-PFP.Mice were injected with cRGD-PLGA-IR780-PFP via tail vein at different times,and blood samples were collected for blood routine and blood biochemical analysis.Results The mean sizes of cRGD-PLGA-IR780-PFP nanoparticles were about(255.83 ± 5.46)nm and the potentials were about(-0.55 0.037)mV.The nanoparticles were regular and uniform in TEM and SEM.Nuclear magnetic resonance hydrogen spectrum(H-NMR)showed that cRGDPLGA has obvious peptide characteristic peaks,which was not seen in PLGA.Confocal microscopy observed the green fluorescence of FITC and the red fluorescence of DiI can merged into orange fluorescence.The connection rate of cRGD measured by flow cytometer is up to 98.06%.Ultraviolet spectrophotometer detected that cRGD-PLGA-IR780-PFP had a strong absorption peak at 780 nm,and the encapsulation rate was(91.93 ± 0.68)% according to the standard curve of IR780.Three kinds of nanoparticles(cRGD-PLGA-IR780-PFP,cRGD-PLGA-IR780 and PLGA-IR780-PFP)were safe to B16 cells by CCK-8 assay.The results of blood routine and blood biochemical indexes showed that cRGD-PLGA-IR780-PFP had no biological toxicity.Conclusion The successfully prepared targeted multifunctional nanoparticles(cRGD-PLGA-IR780-PFP)containing IR780 and PFP has uniform size,good dispersion,high connection rate with cRGD and good biosafety,laying a foundation for further multimodal imaging and systemically photothermal therapy.PART 2 THE STUDY OF IR780 LOADED MULTIFUNCTIONAL NANOPARTICLES ENHANCING MUTIMODAL IMAGINGObjective To investige the ability of IR780 loaded multifunctional nanoparticles enhancing US/ PA/NIRF imaging in vitro and in vivo.Methods The experiment of in vitro ultrasound imaging was divided into different groups with various concentrations of c RGD-PLGA-IR780-PFP,group of c RGD-PLGA-PFP and group of saline.All groups were irradiated with 808 nm laser to observe ultrasound images at different time points,and the phase transition process of targeted nanoparticle was observed by optical microscopy.In vitro PA imaging and NIRF imaging were respectively studied to observe the abilities of c RGD-PLGA-IR780-PFP with different concentrations conducting PA/NIRF imaging by using PA imaging system and Xenogen IVIS Spectrum imaging system,and the corresponding PA and fluorescence values were recorded.In vivo multimodal imaging experiments were divided into I: c RGD-PLGA-IR780-PFP(targeted group),II: PLGA-IR780-PFP(non-targeted group)and III: Saline(control group),of which the PA imaging is conducted through injecting I-III by tail vein to melanoma B16 cells tumor-burdened mice,NIRF imaging experiments are injected with I and II.PA or NIRF imaging of mice in all groups were conducted at different time points(0,0.5,6,24 h)and the corresponding PA and fluorescence values were recorded.Besides,after 24 h of NIRF imaging,in vitro major organs and tumors were imaged by Xenogen IVIS Spectrum imaging system and the corresponding fluorescence values were recorded.In vivo US imaging experiment was conducted by intravenous injected I-III to melanoma B16 cells tumorburdened mice.After 6 h,the tumors were irradiated with 808 nm laser.US imaging was performed on mouse tumors and the gray scale values of tumor areas were recorded.Results c RGD-PLGA-IR780-PFP enhanced US/PA/NIRF imaging in vitro.The higher the concentration was,the higher the corresponding ultrasonic gray order value would be.The temperature of nanoparticles reached the peak values after irradiating for 5 min by 808 nm laser.And after irradiation for 10 min,the gray values of each group decreased because of the completion of phase transformation,which could be detected by optical microscopy.In vitro PA imaging showed that the PA values were positively related with the concentrations of nanoparticles.NIRF imaging in vitro showed that the fluorescence values gradually increased with the rise of the nanoparticle concentration(1-20 mg/ml),and decreased when the concentration exceeded 20 mg/ml.PA imaging and NIRF imaging in vivo showed the PA and fluorescence values of tumors in target group reached to the highest after injection of nanoparticles for 6 h,and decrease after 24 h.The non-target group and saline group within 24 h PA and fluorescence enhancement was not obvious.The fluorescence intensities of in vitro organ and tumors in target group were higher than in non-target group after injection for 24 h.In vivo US imaging showed that 6 h after injection of nanoparticles into tumor-bearing mice,the US imaging was significantly enhanced in the target group,but not in the non-targeted group and the saline group.Conclusion The prepared c RGD-PLGA-IR780-PFP nanoparticles can enhance in both in vitro and in vivo US/PA/NIRF imaging.The phase transformation under laser irradiation can enhance the US imaging in vivo and in vitro.The strong absorptions of IR780 in near infrared region made targeted multifunctional nanoparticles strengthen PA and NIRF imaging in vivo and in vitro.And targeted nanoparticles can mostly arrive at the tumor position after intravenous injection for 6 h.PART 3 THE STUDY OF IR780 LOADED MULTIFUNCTIONAL NANOPARTICLES ENHANCING ENHANCED PHOTOTHERMAL THERAPYObjective To investigate the ability of IR780 loaded multifunctional nanoparticles(c RGD-PLGA-IR780-PFP)enhancing PTT in vivo and in vitro.Methods c RGD-PLGA-IR780-PFP nanoparticles were diluted into groups with different concentrations.The temperature changes of c RGDPLGA-IR780-PFP were detected by infrared thermal imager under irradiation by 808 nm laser(2 W/cm2),and the temperature changes of nanoparticles(5 mg/ml)under irradiation with various powers of 808 nm were also recorded.The experiments of in vitro PTT were divided into I: c RGD-PLGA-IR780-PFP,II: PLGA-IR780-PFP,and III: c RGD-PLGAIR780.The toxic effects of different nanoparticles co-cultured with B16 cells with or without 808-nm laser irradiation were measured by CCK-8 assay.The cell activities of I-III with or without 808 nm laser irradiation was studied by confocal microscopy,in which saline was control.In vivo PTT was performed on melanoma B16 cell bearing mice,of which the experiments were divided into 1: c RGD-PLGA-IR780-PFP + NIR,2: PLGA-IR780-PFP + NIR.3: c RGD-PLGA-IR780 + NIR,4: saline + NIR,5: c RGD-PLGA-IR780-PFP,and 6: saline.All mice in each group were intravenous injected with the same concentration of nanoparticles,and tumors of 1-4 groups were irradiated by 808 nm laser after that.The whole irradiation processes were monitored by near infrared thermal imager.After treatments there were 14 days for us to obverse and record the changes of tumor sizes and body weights of mice.One day after PTT,the H&E staining,TUNEL and PCNA detections were conducted to tumors.Results The maximum temperatures of c RGD-PLGA-IR780-PFP irradiated with the same power of laser were positively related with concentrations of nanoparticles.And at the same concentration,the higher the power of laser is,the higher the temperature is.Cytotoxicity tests in vitro showed that different nanoparticles of I-III were safe for B16 cells at 0-1 mg/ml concentration.In vitro PTT experiments indicated that the therapeutic effect of targeted nanoparticle with laser irradiation was stronger than other groups,which showed PFP could enhance photothermal effect of nanoparticles.In vivo PTT showed that the temperature of nanoparticles with laser irradiation rised to the highest than other groups.And the growth of tumors in this group was significantly inhibited during the observation period.The H&E staining,PCNA and TUNEL detection verified the strongest photothermal effect of targeted nanoparticles with laser irradiation.Conclusion The photothermal effect of c RGD-PLGA-IR780-PFP under laser irradiation is very strong in vitro and in vivo experiments,and the temperatures of nanoparticles showing concentration dependences and laser power dependences.The photothermal effect of the nanoparticles can be significantly enhanced when the nanoparticles undergo phase transition.In vivo systemically photothermal treatments can significantly inhibit the growth of tumors.