Targeted ShRNA-loaded Liposome Complex Combined With Ultrasound For Blood Brain Barrier Disruption And Suppressing Glioma Growth

PART?THE PREPARATION AND CHARACTERIZATION OF SHRNA-LOADED LIPOSOME COMPLEX Objective1 To prepare the microbubble?MB?,liposome?lipo?,targeted liposome?lipo-NGR?and sh Birc5-loaded targeted liposome?shBirc5-lipo-NGR?respectively,and prepare the microubble-shBirc5-loaded targeted liposome?MB-sh Birc5-lipo-NGR?eventually,and characterize of all kinds of product accordingly.2 To study the gene loading capacity and DNA se?protection assay.Methods1 The shBirc5 plasmid was extracted by using high purity plasmid extraction kit.2 MB were prepared by mechanical vibration method,lipo were prepared by sequential extrusion,the connection of lipo and NGR were prepared by carbodiimide method,MB and shBirc5-lipo-NGR were bound by biotin-avidin method.Optical microscope and fluorescence microscope were used to observe and count the products,dynamic light scattering was used to detect the size and potential of each product.3 gene loading capacity and DNA se?protection assay were using agarose gel electrophoresis experiment.Result1 we successfully prepared MB,lipo,lipo-NGR,shBirc5-lipo–NGR and MB-shBirc5-lipo–NGR.The particle size and zeta potential of shBirc5-lipo-NGR were 241.4±56.38 nm and 10.9±3.76 mV,MB-shBirc5-lipo-NGR were 2905±377.8 nm and 14±4.18 mV,Large of shBirc5-lipo-NGR is closely connected to the MB by fluorescence microscope.2 The shBirc5 was completely combined until the ratio of lipo:shBirc5was approximately 5:1.The shBirc5 escaped from DNAse?degradation after combination with lipo-NGR.ConclusionWe successfully prepared shBirc5-lipo–NGR which has high gene loading capacity and good DNAse?protection,and ultimately successfully wprepared MB-shBirc5-lipo–NGR.PART ? TARGETED SHRNA-LOADED LIPOSOME AOMBINED WITH ULTRASOUND INFLUENCE ON C6 CELLS.Objective1 Explore influence on C6 cells'viability when MB-shBirc5-lipo–NGR combined with different ultrasound parameters,clarify the optimal parameters of ultrasound while combined with MB-shBirc5-lipo–NGR for gene delivery.2 Confirm the CD13 mediated targeting to C6 cells of MB-shBirc5-lipo–NGR when combined with ultrasound,and explain the high efficiency of gene transfection.3 Confirm MB-sh Birc5-lipo-NGR combined with ultrasound can targeted decrease Birc5 gene expression of C6 cells.Methods1 Detecting cell vability after MB-shBirc5-lipo–NGR combined with different ultrasound parameters using CCK8,the parameters whose cell vability>75%were selected for further gene transfection efficiency testing.2 The CD13 siRNA was transfected into C6 cell by adenovirus to obtain CD13?-?C6 cells;lipo were dyed by DiI before being prepared to MB-shBirc5-lipo-NGR and MB-shBirc5–lipo,the the products were combined with ultrasound to C6 cells and CD13?-?C6 cells,at last the lipo were observed by using conclusion laser scanning confocal microscope?CLSM?.3 determine the transfection efficiency of C6 cells with different situation by flow cytometry technique,explain the high transfection efficiency of MB-shBirc5-lipo-NGR combined with ultrasound.4 detecting the Birc5 mRNA and protein expression using RT-PCR and WB respectively,and detecting C6 cells apoptosis rate using flow cytometry technique.Result1 Cell viability was lower than 75%in groups exposed to more than 2W or for durations exceeding 60 s.The Optimal ultrasound parameters was30 s/1.0 W,the shBirc5 gene transfection rate reached 34.55%,the cell activity was 82.57%.2 The liposomes in group of MB-shBirc5-lipo–NGR combined with ultrasound on C6 cells was more than the group of MB-shBirc5–lipo combined with ultrasound on C6 cells and the group of MB-sh Birc5-lipo–NGR combined with ultrasound on CD13?-?C6 cells.3 The highest transfection efficiency was observed in the FUS-aided MB-shBirc5-lipo-NGR group,this efficiency was almost 3 times higher than with a commercially available delivery agent,lipofectamine 3000.The efficiency was higher than the MB-shBirc5-lipo group's efficiency of21.26%and MB-shBirc5-lipo–NGR on CD13?-?C6 cells additionally.4 Birc5 mRNA and protein were apparently reduced in the FUS-aided MB-shBirc5-lipo-NGR group compared with the control group.Both mRNA and protein were slightly reduced in the ultrasound group and MB-shControl-lipo-NGR group,and there was no obvious difference when compared with control groups.The FUS-aided MB-shBirc5-lipo-NGR group had an apoptosis rate of up to 38.84%,which exceeded that of all othergroups.Ultrasound-aidedMB-shBirc5-lipoand MB-shBirc5-lipo-NGR group were 19.89%and 18.70%respectively.Conclusion MB-sh Birc5-lipo-NGR combined with ultrasound has the ability to promote gene transfect into C6 cells,sh Birc5-lipo-NGR showed well target on C6 cells which was mediated by CD13.MB-shBirc5-lipo-NGR combined with ultrasound can targeted decrease the Birc5 mRNA and protein expression,and promote C6 cells apoptosis ultimately.PART?TARGETEDSHRNA-LOADEDLIPOSOME COMBINED WITH ULTRASOUND FOR BLOOD BRAIN BARRIER DISRUPTION AND SUPPRESSING GLIOMA GROWTH IN SD RATS.Objective1 Clarify the MB-shBirc5-lipo-NGR dose and ultrasound duration time when combined use them for BBB disruption.2 Clarify MB-shBirc5-lipo-NGR combined with ultrasound can promote shBirc5-lipo-NGR through BBB and target to tumor model,and the gene can express successfully.3 Explore the impact of MB-shBirc5-lipo-NGR combined with ultrasound on SD rat orthotopic glioma model.4 Study the toxic effect of MB-shBirc5-lipo-NGR combined with ultrasound on SD rats.Methods1 Adopting different MB-shBirc5-lipo-NGR dose and ultrasound duration time to open the BBB,animals were subsequently injected with Evans blue?EB?,through the observation of EB dye leakage to confirm BBB disruption.And through HE staining to clear erythrocyte leak out.2 Establish Rat orthotopic C6 glioma model;prepare MB-shBirc5-lipo and MB-sh Birc5-lipo–NGR after lipo dyed by DIR,then the products were injected into rats though tail vein,the liposomes were showed by IVIS Lumina series?in vivo at 2h and 24h later.3 Observe the GFP expression in normal brain tissue and tumor tissue by CLSM after shBirc5 carrying gene expression.4 40 rats with orthotopic C6 glioma were randomly divided into 5groups:Group 1,the control group,was treated with 1 ml of saline;Group2 was treated with 1 ml of saline before FUS exposure;Group 3 was treated with 1 ml of MB-sh Control-lipo-NGR;Group 4 was treated with 1ml of MB-shBirc5-lipo-NGR;and Group 5 was treated with 1 ml of MB-shBirc5-lipo-NGR immediately before FUS exposure.The tumor growth of 3 rats in every group were monitored longitudinally with a 7.0 T MRI scanner at 12,17,22 days after inplantation.Calculated tumor size and growth rate,record survival time of each rat.5 Health SD rats were randomly divided into MB-shBirc5-lipo-NGR combined with ultrasound and saline groups,record their weight,eating and activity.Harvest the main organs for HE staining at 16th day after processing to evaluate the toxicity of MB-shBirc5-lipo-NGR.Result1 The BBB of rats were opened and no obvisous erythrocyte leak out with the condition that MB-shBirc5-lipo-NGR dose was 4 x 10⁷,ultrasound duration time was 3 min.There was a little of erythrocyte leak out 24 h after irradiation and this leak out was absorbed in 48h by HE staining.2 we were successfully established Rat orthotopic C6 glioma model confirmed by MRI T2.The red fluorescence was observed in FUS-aided MB-shBirc5-lipo-NGR group was 8.5 fold higher than FUS-aided MB-shBirc5-lipo group at tumor site 2h after irraditon.The red fluorescence was observed only in FUS-aided MB-shBirc5-lipo-NGR group at tumor site 24h after irraditon.3 Compared with the MB-shBirc5-lipo-NGR group that did not receive FUS,the FUS-aided MB-shBirc5-lipo-NGR group exhibited significant green fluorescence within the tumor region,but no green fluorescence in normal tissue.Green fluorescence was not observed in controls or the ultrasound-only group within either tumors or normal brain tissue.4 The control group exhibited an average tumor volume and growth rate 10 times greater than that of the FUS-aided MB-shBirc5-lipo-NGR group on the 12^th and 17^th day,while the control group with the FUS group,FUS-aided MB-shControl-lipo-NGR group and the MB-shBirc5-lipo-NGR alone group has no significant difference.At 22th day,FUS-aided MB-shBirc5-lipo-NGR tumor volume is significantly less than any other group,but the tumor growth rate between 17 to 22 days was no statistical difference with other groups.The median survival times of the control,FUS,FUS-aided MB-sh Control-lipo-NGR,MB-shBirc5-lipo-NGR and FUS-aided MB-shBirc5-lipo-NGR groups were 21,24,23.5,27,38 days,respectively.5 There were no no obvious difference in weight and weight growth rate bettwen two groups?p>0.05?.The HE staining of the heart,liver,spleen,kidney and lung tissue showed no obvious pathological changes.ConclusionMB-sh Birc5-lipo-NGR combined with ultrasound can disrupt the BBB,targete to tumor cells,promote gene expression,has obvious inhibitory effect on tumor growth,can extend tumor-burdened rats'survival time,and does not produce toxic effects.