The Preparation Of CNA35? Collagen Targeted Liquid-gas Phase Transformation Fluorocarbon Nanoparticles And Study Of Detection For Targeting Myocardial Fibrosis In Vitro

PART? PREPARATION AND CHARACTERISTICS OF PFP NPS AND CNA35-PFP NPSObjectives To prepare liquid-gas phase transformation nanoparticles with targeted type I collagen,and detect basic characteristics of the fluorocarbon nanoparticles.Methods lipids wrapped phase-transformation type liquid perfluorocarbon nanoparticles(PFP NPs)with shell of lipids and kernel of PFP.PFP NPs were prepared by filming-rehydration and acoustic-vibration methods.The morphology,distribution,particle size and zeta potential of PFP NPs were measured to screen out the optimum conditions for preparing fluorocarbon nanoparticles;The specific antibody CNA35 and PFP NPs were linked by carbodiimide method to prepare targeted fluorocarbons nanoparticles(CNA35-perfluoropentane nanoparticles,CNA35-PFP NPs),and the basic properties of CNA35-PFP NPs were tested.The connectivity of CNA35 and PFP NPs was qualitatively observed by confocal laser scanning microscopy(CLSM)and quantitatively analyzed by flow cytometry.Results PFP NPs and CNA35-PFP NPs prepared by two pulses of acoustic vibration at the ratio of 1:20 between the PFP and the lipid suspension were uniform in size and good in dispersion.The NPs were milky white in solution.The NPs were observed to be spherical or punctate by optical and fluorescence microscopes.Transmission electron microscopy showed that the diameter of NPs was about 250 nm.The particle sizes and zeta potentials of PFP NPs and CNA35-PFP NPs measured by Malvern laser particle size potentiometer were(255.86±3.13)nm and(295.62 ±4.19)nm,(-1.73±0.46)mV and(1.79±0.13)mV,respectively.CLSM showed that the green fluorescence of CNA35 and the red fluorescence of PFP NPs were effectively combined.The results of flow cytometry showed that CNA35 and PFP NPs were relatively high and connection rate was about 86%.Conclusion Qualified PFP NPs and CNA35 targeted fluorocarbon nanoparticles(CNA35-PFP NPs)were successfully prepared.CNA35 is a type I collagen specific antibody,which laid the foundation for subsequent in vitro target-seeking experiments of myocardial fibrosis.PART? THE STUDY OF PHASE-TRANSFORMATION AND ULTRASOUND IMAGING IN VITRO OF CNA35 TARGETED FLUOROCARBON NANOPARTICLESObjectives To prepare a novel liquid-gas phase-change targeted type I collagen fluorocarbon nanoparticles—CNA35-PFP NPs,and to investigate the suitable conditions under which heat and low intensity focused ultrasound(LIFU)irradiation could cause the liquid-gas phase transition of the new type of fluorocarbon nanoparticles in vitro,and the development efficiency of the new type of fluorocarbon nanoparticles was observed by diagnostic ultrasound.Methods CNA35-PFP NPs were heated in water bath at different temperatures(37?,40?,45? and 50?)for 1 min,5 min and 10 min,respectively.In addition,CNA35-PFP NPs were irradiated by LIFU at different power(1W/cm2,2W/cm2,3W/cm2,4W/cm2)for 1 min,2min,3min and 4min,respectively.The phase transition conditions of CNA35-PFP NPs caused by heating and LIFU irradiation were observed by optical microscopy and diagnostic ultrasound.The liquid-gas phase transition of CNA35-PFP NPs induced by heating and ultrasonic energy were discussed to screen out the optimum conditions for phase transition.Results Both heating and LIFU irradiation could induce phase transition of CNA35-PFP NPs.It was difficult to observe nanoparticles under a 100-fold microscope before heating and LIFU irradiation.With the prolongation of heating temperature and time in water bath,more and more obvious phase transformation of nanoparticles could be observed under the microscope,the number and volume of nanoparticles increased,simultaneously,ultrasound development efficiency could be enhanced obviously.In addition,the phase transformation of nanoparticles could be promoted and imaging effect enhanced after irradiation with a certain power of LIFU(p < 0.05).We observed that the optimum irradiation intensity of nanoparticles is 3W/cm2 and the irradiation time is 3min to get the best effect ultrasound imaging.Conclusion The CNA35-PFP NPs prepared in our study could undergo liquid-gas phase transition by heating or irradiation with a certain power of LIFU and enhance ultrasound imaging efficiency,which laid a foundation for the diagnosis and treatment of myocardial fibrosis in the future.PART? IN VITRO LOCALIZATION,ULTRASOUND IMAGING AND SEMI-QUANTITATIVE ANALYSIS OF CNA35 I COLLAGEN TARGETED FLUOROCARBON NANOPARTICLESObjectives To investigate the targeted ability of CNA35-PFP NPs on myocardial type I collagen in vitro,and to make a semi-quantitative analysis.Methods The frozen sections(10?m)of myocardial tissue from rabbit model of myocardial infarction and normal rabbit model were prepared respectively,with 5 group per each.FITC-CNA35 was obtained by labeling CNA35 with fluorescein isothiocyanate(FITC).FITC-CNA35 was used to stain the frozen sections of infarction group and control group,observed by CLSM.The same frozen section was stained with Picric acid-Sirius Red(PSR),and the images were collected by optical microscope,and compared with those stained with FITC-CNA35 to verify the targeted ability of CNA35 to collagen fibers;The fluorescent CNA35 targeted fluorocarbon nanoparticles(FITC-CNA35-PFP NPs)were obtained by connecting FITC-CNA35 with PFP NPs by carbodiimide method.The frozen sections of the two groups were stained with FITC-CNA35-PFP NPs and DNA fluorescent dyes(DAPI).The staining results were observed by CLSM.Afterwards,the images were collected and compared with the pathological sections of myocardium in infarction group and normal group,including hematoxylin-eosin(HE),Masson staining and PSR staining.The percentage of collagen fibers in Masson staining,PSR staining and fluorescence staining images was semi-quantitatively analyzed by using analysis software Image J to verify the target-seeking ability of CNA35-PFP NPs in vitro.Results A large amount of green fluorescence was observed in the fluorescence images of infarction group,while no obvious green fluorescence was observed in normal group,where the location of green fluorescence was basically the same as that of collagen fibers stained by PSR.The results of FITC-CNA35-PFP NPs and DAPI staining were compared with those of HE,Masson and PSR staining.It was found that the CNA35-PFP NPs represented by green fluorescence could congregate in a large number of collagen-rich areas,and a consistent result can be obtained by semi-quantitative analysis.Conclusion CNA35-PFP NPs could bind to myocardial type I collagen fibers and accumulated in abundance with the deposition of myocardial collagen fibers.Therefore,CNA35-PFP NPs had a great targeted ability in vitro,which could provide a basis for subsequent in vivo targeting research.