| Purpose Develop a NBs which is fabricated by thin film hydration protocol and si RNA combination by biotin and streptavidin system,and predict NBs carrying si RNA combined with UTMD would improve si RNA transfection efficiency,to provide experimental support for NBs carrying si RNA combined with UTMD as a noninvasive treatment of glioma.Method 1.DSPE-PEG?2000?-biotin and DPPC were dissolved in 2ml chloroform.Then rotary evaporation was performed.1ml hydration liquid was added.Then,placed the bottle into an incubator shaker to form liposomal film suspension.The suspension was equally divided into two tubes with plastic caps.The air in the sealed tubes was replaced with C3F8 gas using syringe.Finally,every tube was oscillated for 45 s in an amalgamator.The bubbles in each tube was separately diluted to 8 ml by PBS.The MBs used in the study was fabricated.The dynamic light scattering?DLS?was used to measure the particle sizes of the NBs and MBs.NBs suspension was examined by scanning electron microscopy.The CCK-8 was used to test cytotoxicity of NBs..2.100?l NBs suspension was diluted by RNase-free PBS to 850?l and 100?l streptavidin as 10mg/ml was added into the tube.The tube was oscillated for 30 min.Then,50?l 20?M si RNA suspension was mixed and oscillated again.The air in the tube was replaced by C3F8 gas and kept the tube in the 4? ice box for 90 min.After delamination appeared in the suspension,lower layer clear suspension was draw by syringe and equal amounts of PBS was injected.This step was repeated two or three times.Finally,the NBs-si RNA formed.NBs-SCR and NBs-FAM-SCR were fabricated same way.The particle size and stability of NBs-si RNA were measured by DLS.NBs-si RNA was measured at 1 15,30,45 and 60 min.At each time point,the NBs-si RNA were concurrently transferred to a hemocytometer for counting.NBs-FAM-SCR was observed by fluorescence microscope.And the bare NBs was used as control.3.5 x104 glioma U87 cells were seeded in 12-well plates.The sample suspension was added.Ultrasound exposure parameters were 1 MHz frequency,0.88 W/cm2 intensity and 45 s exposure time.4.The transfection efficiency of FAM-SCR was detected by confocal laser scanning microscopy?CLSM?.There were three groups: NBs carrying FAM-SCR with ultrasound irradiation?NBs-FAM-SCR-UI?,FAM-SCR with ultrasound irradiation?FAM-SCR-UI?and bare FAM-SCR?control?.24 h after transfection,cells were washed by PBS for three times and cell nucleus were stained by DAPI to identify the location of cells.Cells were observed on confocal laser scanning microscopy.5.1)The mRNA expression of target gene was detected by real-time PCR assay 48 h after transfection.The experiment groups were NBs carrying si RNA with ultrasound irradiation?NBs-si RNA-UI?,si RNA with ultrasound irradiation?si RNA-UI?and NBs carrying SCR with ultrasound irradiation?control?.2)The protein expression of target gene was detected by Western blot analysis.The experiment groups were similar to above.6.Annexin-V was used to detect cell apoptosis.The groups were as former.2)CCK-8 was applied to evaluate the cell viability after transfection.7.The glioma in vivo model was established by the subcutaneous injection of 5 × 106 U87 cells into the right back area of athymic nude mice.8.After isoflurane anesthesia,mouse were injected by NBs-siRNA suspension?200?l?through the tail vein.15 s after the enhancement appeared,targeted destruction microbubbles ultrasound that was 1 MHz frequency,1.5 W/cm2 intensity and 1min expose time was implemented.The enhanced tumor images before and after ultrasound irradiation were recorded?UI?+?group?.The enhanced tumor images without ultrasound irradiation at same time point was used as control?UI?-?group?.The gray scale intensity of digital clips and images were obtained and analyzed.2)Di I-NBs-si RNA and Di I-MBs-si RNA?200?l?were separately injected into nude mouse bearing glioma xenograft tumors.The tumors were separated to prepare sections.Finally,CLSM was used to observe the tumor sections.3)The mouse were sacrificed and tumors,livers,and spleens were harvested 10 min,30min,60 min,6h,12 h and 24 h after Di I-labeled NBs-si RNA injected through the tail vein.The fluorescence images of tumors and organs were obtained and analyzed via the IVIS Lumina II system.9.The mouse for in vivo experiment were randomly separated into four groups: NBs carrying si RNA with ultrasound irradiation?NBs-si RNA-UI?,MBs carrying si RNA with ultrasound irradiation?MBs-si RNA-UI?,NBs carrying si RNA without ultrasound irradiation?NBs-si RNA?,and NBs carrying SCR with ultrasound irradiation?NBs-SCR-UI?.200?l NBs carrying si RNA or SCR suspensions were injected through tail veins.Then,the tumor were exposed to ultrasonic irradiation.1)The volume of tumor was calculated.Survival rate of mouse of the four groups was analyzed.2)After the mouse sacrificed,tumors were separated to prepare paraffin sections and Hematoxylin/eosin?H&E?staining and TUNEL assay were performed.Results 1.The average particle size of the bubbles were measured by DLS.The average particle size of NBs is 625.4 ± 63.8nm?n=3?and that of MBs is 2355.8 ± 512.8nm?n=3?.In the SEM micrographs,the NBs was observed as un-aggregated hollows with about 400-800 nm particle size which is corresponding with the size distributions measured by DLS.The cytotoxicity curve of phospholipid concentration was detected by CCK-8.After statistical analysis,it would be observed that when the concentration increased to 10 ?g/ml obvious cytotoxicity appeared.2.The stability of NBs-siRNA at room temperature was evaluated by particle size and concentration.The average particle size of NBs carrying si RNA stored separately at 25 °C for 1,15,30,45 and 60 min were 688.23 ± 61.4nm?n=3?,740.33 ± 54.88nm?n=3?,756.37 ± 51.20nm?n=3?,789.13 ± 62.22nm?n=3?and 870.17 ± 93.09nm?n=3?.Compared with the average particle size of NBs carrying si RNA stored for 1min,there was no statistical difference at each time point.The concentration of NBs carrying si RNA were 88.34×105 ± 6.59×105/ml?n=5?,77.02×105 ± 6.62×105 /ml?n=5?,70.76×105 ± 4.29×105 /ml?n=5?,62.00×105 ± 7.53×105 /ml?n=5?and 50.72×105 ± 6.91×105 /ml?n=5?at 1,15,30,45 and 60 min.The statistical analysis shown that there was significant difference between the concentrations of each time point with that at 1 min.The optical and fluorescence images of NBs-FAM-SCR group and the control group bare NBs were observed.In the NBs-FAM-SCR group,green fluorescence would be observed and its conformity with NBs under optical microscope and fluorescence was great.And no fluorescence was observed on the bare NBs.3.The CLSM was used to evaluate the cell uptake of FAW-SCR.It was shown that cell nucleus could be seen as blue in all groups and green florescence in perinuclear and cytoplasm would be observed in NBs-FAM-SCR-UI group and FAM-SCR-UI group,not in control group.However,compare to FAM-SCR-UI group,green florescence was more and stronger in the NBs-FAM-SCR-UI group.4.Transfection efficiency was assessed in m RNA level by real-time PCR assay and in protein level by western blot analysis.Compared with the control group,the IDH1 m RNA of both NBs-si RNA-UI and si RNA-UI groups decreased.Similar to real-time PCR result,the result of western blot analysis revealed that the IDH1 expression of NBs-si RNA-UI group at the protein level was much lower than si RNA-UI group though the latter being depressed compared with control group.The result of cell apoptosis analyzed by flow cytometry indicated that NBs-si RNA-UI group is higher.Meanwhile,cell viability of NBs-si RNA-UI group was lowest.5.It would be observed that imaging contrast enhancement appeared after NBs-si RNA injection in both UI?+?group and UI?-?group.However,the contrast enhancement of UI?-?group was stronger than the control group UI?+?group after ultrasound irradiation.Significant difference occurred after UI in the two group.6.The CLSM examination of tumor frozen sections was obtained to locate Dil labeled NBs-si RNA.The blood vessels were dyed green,whereas the nuclear were blue.Dil labeled MBs-si RNA would not be found and NBs-si RNA was observed in the extravascular intercellular space.The fluorescence of the liver and tumor increased obviously after Dil labeled NBs-si RNA injection and gradually decreased,meanwhile the spleen maintained a relatively lower level.7.Compared to the other three groups,the tumor volume of NBs-si RNA-UI group was smaller.In comparison,the NBs-si RNA-UI group owned a better therapeutic effect.The histological changes of each group were observed by H&E staining.It would be observed that the NBs-si RNA-UI group owned fewer cells and lower level polymorphism which are the opposite in the other three groups.The TUNEL assays were performed to evaluate apoptosis of tumor.Obviously,cell apoptosis of NBs-si RNA-UI were higher than the other three groups.Conclusion In summary,we fabricated nano-size bubbles,connected it with si RNA by avidin-biotin,and combined NBs-si RNA and UTMD to achieve improvement of si RNA transfection efficiency in vitro.Furthermore,due to its small size,NBs-si RNA would penetrate the tumor blood vessel into intercellular space in vivo and realized the purpose of suppressing tumor growth in vivo.NBs would be used as vector of si RNA delivery for noninvasive treatment of glioma. |
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