Experimental Study On Real-time Imaging Tracking Of Bone Mesenchymal Stem Cells Labelled With Lipid PLGA Nanobubbles

Chapter 1 Preparation and characterization of lipid PLGA nanobubblesObjective To prepare a novel hard shall ultrasound contrast agent,lipid PLGA nanobubbles(LPNs),which with good biocompatibility and can achieve stable ultrasound imaging in animals for a long time for endocytosis of stem cell.Method LPNs were prepared by double emulsion evaporation process and vacuum freeze method with PLGA and lipid as film forming materials.The morphological characterization of LPNs was analyzed by Scanning Electron Microscope and Transmission Electron Microscope.The average diameter and size distribution of LPNs were determined by dynamic light scattering(DLS).Ultrasound imaging performance of pure LPNs were detected by ultrasonic imaging device.Result SEM image revealed that LPNs possessed the high dispersity and well-defined spherical morphology.From the TEM image,a big cavity could be seen in the cores of LPNs.The particle size of the resulting LPNs measured by DLS showed the average diameter of these LPNs was(467.3±19.49)nm.Strong ultrasound contrast signals of these nanobubbles could be observed,the higher nanobubble concentrations would produce the stronger ultrasound contrast signals,showing a good linear correlation(R2 = 0.99).These results suggested that these nanoscale LPNs can function as the excellent ultrasound contrast agents.Conclusion The LPNs were successfully prepared which have regular m,orphology and uniform size.They can produce strong and stable ultrasound contrast-enhanced signal with good acoustic performance.Therefore,LPNs is a new type of ultrasound contrast agent has good prospect.Chapter 2 Construction and ultrasound tracking imaging in vitro and in vivo of bone mesenchymal stem cells labelled with lipid PLGA nanobubblesObjective In this study,we prepared LPNs labelled bone mesenchymal stem cells(BMSCs).It can investigate the effect of LPNs on the proliferation of BMSCs and evaluate the ultrasonic effectiveness of LPNs labelled BMSCs.The aim is to prepare a labelled stem cell with real-time ultrasound imaging function,thus providing a new strategy for stem cell tracking based on real-time ultrasound imaging,paving the way for underlying mechanism investigation and future clinical application of stem cell therapy.Method1.?Bone mesenchymal stem cells were extracted from rat humerus,ulna and radius by whole bone marrow adherent culture method.2?Bone mesenchymal stem cells were incubated with different concentrations of LPNs(50?g/ml,1000?g/ml,200?g/ml,500?g/ml,1000?g/ml,2000?g/ml)for 6h.Confocal fluorescence microscopy was used to detect the endocytosis effect of stem cells on LPNs.The result of CCK-8 cytotoxicity experiment,in vitro ultrasound performance and confocal fluorescence microscopy imaging was combined to select the LPNs co-incubation concentration with the best endocytosis effect.3?2×106LPNs labelled BMSCs were injected subcutaneously into the back of nude mice.Ultrasound imaging of the target area was observed before and after injection.2×106LPNs labelled BMSCs were injected into the subcutaneous tumor parenchyma of nude mice in situ,then observed their ultrasound imaging at different time points(0,1,5 days).4?2×103,2×104,2×105 LPNs labelled BMSCs suspended in 100?l PBS were injected subcutaneously into the back of nude mice,we determine the fewest number of labelled BMSCs which can be detected by ultrasound imaging in vivo.Result1?BMSCs were isolated by method of whole bone marrow adherent.The cells were spindle and have biological characteristics of BMSCs.2?The CCK-8 assay indicated these uptaken LPNs had no significant cytotoxicity to BMSCs since there were no significant viability differences between the labelled cells with control group(without LPNs)(p>0.05).There was a good linear relationship between the ultrasound contrast signal intensity and LPN concentrations used for labelling these BMSCs from 0 to 500?g/ml,but it defines the upper limit after 500?g/ml.Therefore,we used 500?g/ml LPNs to label BMSCs for all subsequent experiments.3?LPNs labelled BMSCs remained the ultrasound contrast imaging up to 5 days in vitro.We injected labelled BMSCs into the subcutaneous tumor parenchyma of nude mice in situ,notably,the implanted BMSCs were serially monitored by ultrasound imaging for 5 days.4?The fewest number of labelled BMSCs which can be detected by ult:rasound imaging in vivo was 2000 LPNs labelled BMSCs.Conclusion In summary,BMSCs can be effectively labeled by LPNs and the labeling does not affect the biological activity of BMSCs,LPNs labelled stem cells can produce strong contrast-enhanced signals which may reduce the background interference signals when used in monitoring stem cell transplantation.It can ensure adequate cell delivered to the target treatment site also enable long-term,real-time stem cells tracking imaging.