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Objective:To fabricate biogenic gas vesicles (GVs)- polyethyleneimine (PEI)-marrow mesenchymal stem cells (BMSCs) and evaluate its potential on stem cell tracking with ultrasound imaging.Methods:GVs were cationized by PEI to fabricate GVs-PEI. The diameter and zeta potential of GVs-PEI were determined. GVs-PEI were co-incubated with BMSCs to obtain GVs-PEI-BMSCs and stem cell uptake was observed by fluorescence microscope. The cell viability of GVs-PEI-BMSCs was verified by cell counting kit-8 (CCK-8) assay were set. Ultrasound imaging was performed on 0, 2, 4 and 6 d in agarose phantom to evaluate ultrasound imaging capability of GVs-PEI-BMSCs group and BMSCs group in vitro. GVs-PEI-BMSCs and BMSCs were injected into quadriceps femoris of SD rats, and ultrasound imaging was performed on 0, 2, 4 and 6 d to evaluate the ultrasound imaging capability in vivo. One-way analysis of variance and independent-sample t test were used to analyze the data. Results:The diameter and zeta potential of GVs-PEI were (383.63±11.55) nm and (18.48±2.20) mV. Plenty of GVs-PEI were observed in GVs-PEI-BMSCs through microscope. When BMSCs were incubated with GVs-PEI in absorbance ( A) 500 nm of 0.5 and 1.0, there were no significant changes in the cell viability of GVs-PEI-BMSCs at 24, 48 and 72 h ( F values: 7.078-11.982, all P>0.05). Compared with BMSCs, GVs-PEI-BMSCs showed better ultrasound imaging capability in vitro in all time points with still significantly different signal at 6 d (634.29±10.78 vs 2 864.51±100.86; t=-121.86, P<0.001). The ultrasound imaging capability of GVs-PEI-BMSCs in vivo was much better than that of BMSCs at each time point with still significantly different signal at 6 d (2 108.02±217.96 vs 267.71±7.87; t=-121.39, P<0.001). Conclusion:GVs-PEI-BMSCs are successfully fabricated with the advantages of significant ultrasound imaging capability, long duration and safety, which provide a brand-new means for stem cells tracking in vivo.
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Objective To fabricate iRGD targeted liposome-microbubble complex containing uPA ( iRGD-LMC) ,and to improve the thrombolytic efficiency and reduce the risk of thrombolysis by iRGD-LMC combining with ultrasound targeted microbubbles destruction ( UTMD ) to release drug into the thrombus site with the help of microbubble cavitation effect . Methods Biotinylated iRGD-MBs were fabricated by thin-film rehydration method .Biotinylated liposomes containing uPA were fabricated by freeze-thaw method and were conjugated to the biotinylated iRGD-MBs surface through biotin-avidin linkage . The iRGD-LMC was subjected to confocal microscopy to determine the particle morphology . The concentration , average diameter and size distribution were determined by particle sizing instrument . The uPA loading efficiency was measured by BCA Protein Assay Kit . Ultrasound imaging was performed using a Vevo 2100 ultrasound imaging system . The iRGD-LMC was irradiated by different ultrasound time and intensity to release drug . Thrombolytic effect in vitro of iRGD-LMC combined with UTMD was observed on the thrombosis model which was extracted from mouse blood . Results iRGD-LMC was successfully prepared . iRGD-LMC was exhibited a well-defined spherical morphology and homogeneous distribution ,like ordinary microbubbles . The concentration of iRGD-LMC was ( 0 .51 ± 0 .03 ) × 109 / ml and average diameter was ( 2 .62 ± 0 .12) μm . Drugs loading efficiency was ( 3878 .5 ± 97 .8) μg uPA per 108 microbubbles . iRGD-LMC could achieve contrast-enhanced ultrasound imaging in vitro . The thrombolytic effect of iRGD-LMC +US group ( 87 .66 ± 1 .69) ％ was the best in vitro ,and had significant difference with others groups ( P <0 .05) ,followed by iRGD-LMC group ( 53 .32 ± 4 .86) ％ and uPA group ( 51 .09 ± 9 .01) ％ ,Compared with PBS group ,US group ( 23 .56 ± 9 .46) ％ had thrombolytic effect . Conclusions iRGD-LMC is successfully prepared ,which has the advantages of high drug loading of liposomes and good acoustic properties of microbubbles . iRGD-LMC combined with UTMD achieves a significant thrombolytic effect in vitro .
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Objective To develop a vulnerable plaque targeting ultrasound contrast agent (UCA) and to evaluate its affinity and imaging performance in vitro.Methods E-selectin receptor-targeting UCA,which conjugated with monoclonal antibody of E-selectin,was prepared with filming-rehydration method and biotin-avidin linkage.The size and distribution of UCA were measured with particle size analyzer,the connectivity condition of microbubbles with E-selectin antibody was also detected with fluorescence analysis.The cytotoxicity from microbubble and ultrasound irradiation was evaluated through cell counting kit-8 (CCK8) assay.The adhesion effect of UCA was assessed after co-incubated with activated mouse endothelial cells (bEnd.3) and compared with that of free antibody intervention group and control group.The imaging performance of UCA at different time points was observed on an ultrasound equipment with a high-frequency transducer.Two-sample t test and one-way analysis of variance were performed to analyze the data.Results E-selectin receptor-targeting UCA was successfully prepared.The cytotoxicity result with CCK8 assay demonstrated the favorable biocompatibility of UCA.The connection amount of UCA on activated bEnd.3 cells ((6.23 ± 0.45) bubbles/cell) was significantly higher than that of the free antibody intervention group ((1.57±0.34) bubbles/cell) and control group ((0.07±0.03) bubbles/cell;F=291.43,P＜0.01).The performance of in vitro ultrasonography at the same time points showed no obvious difference between targeting UCA and control UCA (all t＜0.51,all P＞0.05).Conclusions The prepared E-selectin receptor-targeting UCA has favorable targeting and imaging capabilities.It might be a potentially ultrasound molecular imaging agent for early detection and prognosis evaluation of vulnerable plaque.
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Objective To investigate the expression changes of fractalkine (FKN)in focal cerebral ischemia and reperfusion penumbra,and to explore its variation law and role in the inflammation of cerebral ischemia-reperfusion injury.Methods The cerebral ischemia reperfusionmodel was established by intraluminal thread occlusion in the middle cerebral arteries occlusion (MCAO).FKN protein expression in focal cerebral ischemia and reperfusion penumbra was detected by immunohistochemistry and Western blot.Results The results of immunohistochemistry stain showed that the chemokine FKN was expressed in a low level in the normal group and the sham operation group,and there were no significant differences among the two groups (P＞ 0.05).Compared with the humbers of FKN in normal group (37.03± 6.28) in focal cerebral ischemia and reperfusion penumbra,the expression of FKN in model group was increased after 3 h of reperfusion (48.58±7.29) (P＜0.05),peaked at 24 h (112.08±8.26) (P＜0.05],and then decreased gradually at day 7 after reperfusion,but had no significant difference (40.73 ± 4.02) (P＞ 0.05).FKN was expressed in a low level in the sham operation group (0.527±0.002),then up-regulated after 3 h of reperfusion [(0.598±0.004),P＜0.05],peaked at 24 h [(0.833±0.005),P＜0.05],maintained a high level till 48 h after reperfusion [(0.735±0.002),P＜0.05],and return to baseline level at day 7 after reperfusion [(0.533±0.004),P＞0.05].Conclusions Fractalkine is upregulated after focal cerebral ischemia/reperfusion,and has a dynamical change,which indicates that fractalkine might involve in the inflammatory process after cerebral ischemia-reperfusion injury.