Basic Research On Iron Phthalocyanine-load Molecular Probe In Dual Modal Imaging And Treatment Of Breast Cancer Cell

PART I SYNTHESIS,CHARACTERIZATION AND BIOSAFETY EVALUATION OF AS1411-PLGA@FePc@PFPObjective:To synthesize the phase-transformed nanoparticles loaded with iron phthalocyanine(FePc)targeting breast cancer and testify its physicochemical properties,stability and biosafety.Methods:Phase-change targeting nanoparticles A-FP NPs(AS 1411-PLG A@FePc@PFP)were prepared through two-step emulsification method and carbodiimide method.PLGA loaded with FePc on the shell,PFP was loaded into the core,and aptamer AS 1411,as a targeting probe,was attached to the outside of PLGA.Simultaneously,non-targeting nanoparticles FP NPs(PLGA@FePc@PFP)and targeting blank nanoparticles A-P NPs(AS1411-PLGA@PFP)were also synthesized.The combination between AS 1411 and FP NPs was observed by LCSM,and the connection rate was detected by FCM.TEM was utilized to inspect the morphology of A-FP NPs.Measurement and comparison of A-FP NPs and FP NPs in sizes and potentials were implemented with Malvern particle size analyzer.The absorption spectra of A-FP NPs,A-P NPs and FePc were measured,the standard curve and the encapsulation efficiency of FePc were both calculated.The stability of A-FP NPs was monitored within 2 weeks,and the macroscopic changes were recorded by camera.CCK-8 was used to detect the toxicity of A-FP NPs at different concentrations(0.2,0.4,0.6,0.8,1.0 mg/ml)to cells after incubation for 24 hours.Kunming mice were randomly divided into 5 groups(Control group and 1,3,7 and 14 day group)for blood routine analysis,blood biochemical analysis and HE staining of important organs.Results:AS1411-PLGA?FePc@PFP(A-FP NPs)was successfully synthesized with uniform size and good dispersion according to observation by using the TEM.The connection rate of AS 1411 and PLGA@FePc@PFP was(96.20+2.32)%.The diameters of A-FP NPs and FP NPs were 201.87±1.60 nm and 185.13±5.04 nm,and the surface potentials were-10.67±0.25 mV and-14.30±1.15 mV,respectively.The encapsulation efficiency was(82.26±2.39)%.It can be seen that the absorption spectrum of A-FP NPs is similar to that of FePc.A-P NPs has no obvious absorption peak at 660nm because it is not loaded with FePc.After a 14-days observation,the stability of A-FP NPs in particle size was confirmed.A-FP NPs showed a good biosafety both in vivo and in vivo,and no noticable deaths caused by safety problem occurred.Conclusion:The phase change targeting nanoparticles A-FP NPs,loaded with FePc was successfully synthesized.It has stable structure,good encapsulation efficiency,obvious absorption peak in near infrared band and high biological safety,so it can be used in further experiments.PART ? AS1411-PLGA@FePc@PFP FOR PHOTOACOUSTIC/ULTRASOUND DUAL MODAL IMAGING IN VITRO AND IN VIVOObjective:To explore the optical droplet vaporization(ODV)ability and PA/US dual modal imaging ability of A-FP NPs in vivo and in vitro.Methods:The A-FP NPs was irradiated by near-infrared laser and the phase transition of it was observed under light microscope.The most suitable optimal activation wavelength of the particles was confirmed by full wavelength scan.A-FP NPs in different concentrations were added into the gel model to perform in vitro PA imaging,A-P NPs was set as the control group.The PA imaging ability of A-FP NPs was observed by PA imager,and the PA signal intensity was analyzed.Besides,US/CEUS imaging abilities were observed after irradiation of 660nm laser,and after that the signal intensity values of the regions of interest were delineated.Established female tumor-bearing mouse models were randomly divided into 2 groups,and A-FP NPs and FP NPs were injected through the tail vein,respectively.In vivo PA images were performed to both groups.Images and the corresponding signal values were recorded at different time points(pre,3h,6h,12h,24h).6 hours after the injection of A-FP NPs and FP NPs through the tail vein,660 nm laser was utilized to irradiated tumor site.The CEUS signals were inspected and the corresponding intensity values were measured.Results:Under light microscope,it was observed that A-FP NPs changed from liquid-gas phase to microbubble after laser irradiation,but no phase transition was observed in A-P NPs.In vitro PA/US imaging,the signal intensity of A-FP NPs increases with the increase of concentration.In vivo PA/US imaging experiment showed that the signal intensity of the targeting group reached the highest at 6 h.Compared with the non-targeting group,the signal intensity of the targeting group was higher than that of the non-targeting group,and the signals lasted for longer time than that of non-targeting group.Conclusion:A-FP NPs showed great PA/US imaging effects in vivo and in vitro,which allows A-FP NPs significantly beneficial for imaging monitoring and guidance of therapy.PART ? AS1411-PLGA@FePc@PFP FOR PHOTOTHERMAL TREATMENT OF BREAST CANCER CELLSObjective:To investigate the photothermal transformation ability,photothermal stability,in vitro targeting ability towards human breast cancer MCF-7 cells and the effect of PTT.Methods:PTT of A-FP NPs was explored by using different concentrations of A-FP NPs,the best concentration of PTT in vitro was found.Photothermal stability of the particles was also examined during the process.Targeting ability of A-FP NPs towards MCF-7 cells was observed through CLSM.Irradition of NIR 660nm laser was performed to MCF-7 cell culture dishes co-cultured with A-FP NPs as PTT.Survival status of MCF-7 cells was evaluated after CAM/PI co-staining through CLSM,whereas apoptosis rate was detected by FCM.Results:A-FP NPs have a good performance of targeting in vitro.Partial targeting was achieved one hour after co-incubation,while great enrichment was noticed at the fourth hour.Besides,nanoparticles were proved to have good photothermal conversion ability,which allows faster heating at a lower concentration and effective lethality towards breast cancer cells using thermal effect.The apoptosis rate reached 87.86±4.27%.It has good photothermal stability and made repetitive therapy possible.Conclusion:Strong photothermal conversion ability,great photothermal stability and killing effect on MCF-7 cells of A-FP NPs grant them potential further research value of realizing integration of therapy and diagnosis,which provides treatments of tumors with more possibilities.