| ObjectiveGastric caner(GC)is one of the most common malignant tumor of digestive tract and the second leading cause of cancer death worldwide.To improve survival rate of GC patients,the diagnosis of early GC should be enhanced to provide more treatment chances to patients.For the screening of early gastric cancer without obvious clinical symptoms,contrast-enhanced ultrasound imaging,as a non-invasive and non-radiological imaging diagnosis method,is an important auxiliary diagnostic method.Currently,contrast-enhanced ultrasound imaging combined with molecular imaging technology has greatly improved the specificity and sensitivity of ultrasound imaging of tumor sites.The core of ultrasound molecular imaging is the targeting function provided by the specific binding of receptors and ligands.Studies have shown that the integrin family?v?~3 is highly expressed on the surface of gastric cancer cells,and its specific ligand RGD peptide(Arg-Gly-Asp,arginine-glycine-aspartic acid)has the potential to provide a molecular basis for targeted imaging of GC.In addition,liquid fluorocarbon materials can improve the contrast agent's EPR effect and echo intensity through liquid-gas phase transition in ultrasonic molecular imaging due to their unique advantages of sonogenic phase transition.Therefore,in this study,polymer nanoparticles(RGD-PFH-NPs)modified with RGD peptide and encapsulating perfluorohexane(PFH)were prepared to act as ultrasound contrast agent.Then its basic characteristics,targeting ability were investigated,meanwhile,low intensity focused ultrasound(LIFU)was applied for ultrasound imaging in vivo and vitro.Methods 1.The PFH-NPs was prepared by double emulsification,and then the RGD peptide was attached to the surface of PFH-NPs by carbon diimide method.2.(1)The basic morphology and dispersion of RGD-PFH-NPs were observed by optical microscope.Mean particle size and potential of RGD-PFH-NPs were measured by Malvern Laser Particle Size Meter,and particle size changes were measured for 7consecutive days to investigate its storage stability.The binding rate between RGD peptide and PFH-NPs was quantified by flow cytometry.Phase transition of RGD-PFH-NPs in vitro was investigated by heating plate.(2)CCK-8 assay was used to evaluate the cytotoxicity of RGD-PFH-NPs and the damage effects of different LIFU energies on cells.3.(1)The targeted binding effect of RGD-PFH-NPs on human gastric cancer cell line MGC803 was observed by laser scanning confocal microscope(LSCM).(2)Esaote Mylab 90 ultrasonic diagnostic instrument was used to observe the in vitro ultrasonic imaging ability of RGD-PFH-NPs in agarose gel model and investigate the changes of echo intensity before and after its combination with LIFU.DFY software was uesd to quantify each sound intensity value.4.(1)The tumor-bearing nude mouse model was established by subcutaneous injection of MGC803 cells into the back of nude mice.RGD-PFH-NPs was injected into the tail vein of tumor-bearing nude mice,and the main organs were removed 24 hours later and 7 days later for H&E staining to observe the damage effect of RGD-PFH-NPs on various tissues in vivo.(2)Esaote Mylab 90 ultrasonic diagnostic instrument was used to observe the changes of ultrasound imaging of tumor sites in tube-bearing nude mice before and after injection of different nanoparticles and with or without LIFU,to investigate the imaging capability of contrast agents in vivo.Then the image duration of RGD-PFH-NPs and Sonovue was compared.DFY software was used to quantify each sound intensity value.(3)The aggregation of nanoparticles in tumor tissues and main organs of tumor-bearing nude mice in the targeted group and the non-targeted group were observed by LSCM respectively to investigate the tumor targeting ability and tissue distribution of RGD-PFH-NPs in vivo.Results 1.(1)The prepared PFH-NPs and RGD-PFH-NPs samples were all opalescent suspension,which presented uniform dispersion observed by light microscope and a spherical shape and uniform size observed by transmission electron microscope(TEM)with a core-shell structure.The average particle size was(259.3 42.6)nm,Zeta potential was(-17.6 5.4)mV.No significant change in particle size within 7days represented a good stability of RGD-PFH-NPs.Most fluorescence of the RGD peptide and PFH-NPs coincided with each other observed by LSCM,and the binding rate was 89.13%measured by flow cytometry.When heated to 70?,RGD-PFH-NPs was observed apparent phase transformation,and when heated to 80?,the particle size of RGD-PFH-NPs increased to(1042.4+227.5)nm.(2)CCK-8 assay showed that RGD-PFH-NPs at different concentrations did no significant harm to cell survival rate,and the survival rate of cells irradiated with different LIFU intensity also showed no significant decrease,indicating that RGD-PFH-NPs and LIFU had no significant toxic and side effects on cells.2.(1)In two-dimensional ultrasound mode and contrast enhancement mode,the contrast echo intensity of RGD-PFH-NPs increased as the LIFU time prolonged,and reached the peak at 5 min.The phase transition of nanoparticles was gradually enhanced with the extension of LIFU time according to images of optical microscope.(2)Images of CLSM showed that the number of nanoparticles surrounding MGC803 cells in the targeted RGD-PFH-NPs group increased with time,while that in the non-targeted PFH-NPs group was negligible within 3 h.The binding rates of nanoparticles and MGC803 cells in the two groups were 82.59%and 2.96%respectively.3.(1)Tumor-bearing nude mice injected with RGD-PFH-NPs and then irradiated by LIFU at the tumor site,as a result,the ultrasound imaging effect significantly enhanced while the same agent administrated to mice without LIFU at the tumor site didn't offered any enhancement in ultrasound imaging.In addition,in the group of PFH-NPs with LIFU at the tumor site,no obvious enhancement of ultrasound imaging was observed.All phenomenons illustrated that RGD-PFH-NPs could not only targeted gathered at the tumor site,also could be stimulated by LIFU to enhance ultrasound imaging.(2)The enhanced ultrasound imaging effect of RGD-PFH-NPs at tumor site was lower than that of Sonovue,but the sustained enhancement was more than 60 min,which was significantly longer than that of Sonovue.(3)Images of CLSM showed that both RGD-PFH-NPs and PFH-NPs were mainly concentrated in liver and spleen.RGD-PFH-NPs accumulated in tumor parenchyma,while only a small number of PFH-NPs were accumulated,indicating that the nanoparticles accumulation in the targeted RGD-PFH-NPs group was significantly higher than that in the non-targeted PFH-NPs group(P<0.05).ConclusionTheconstructednanoparticlesRGD-PFH-NPs,providing contrast-enhanced ultrasonic imaging and excellent targeting ability to human gastric cancer cells MGC803 and gastric cancer tissue,is expected to become a new type of gastric cancer targeted ultrasound contrast agent. |
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