Study Of Gene Transfection Enhancement By Ultrasound Targeted Microbubble Destruction

Developments of techniques in molecular biology have led to the progress of genetherapy which is mainly dependent on the development of the methods of gene delivery.Meanwhile,noninvasive delivery systems that could target specific anatomical sites wouldbe hotspot for gene therapy and would be valuable clinical tools.Viral vector has beendeveloped as highly efficient methods for gene delivery to a variety of tissues,but thesafety of this kind of delivery method and immune responses are still concerned in theclinical applications.The effectiveness of nonviral gene vectors for delivering genes intocells has attracted a great deal of attention in recent years.Nonviral vectors are safer,butthe transfection efficiencies are too low to be of clinical value.Among non-viral techniques,ultrasound-targeted microbubble destruction (UTMD)has evolved as a new promising toolfor organ-specific gene delivery in in vitro and in vivo targeting delivery via a processwhich is called sonoporation,allowing for the macromolecule transfering into the cellsefficiently.To make such technology suitable to the clinical setting,parameters of UTMDtechnique should be optimized systematically.Moreover,we assessed whether the novelcombination of UTMD and polyethylenimine (PEI)was available and useful tool for genedelivery and transfection.Furhtermore,a detailed study of the combination of UTMD andshRNA targeting human Survivin gene in vitro and in vivo was carried out in this work andelucidated the effects of gene silencing cell apoptosis and proliferation,laying thefoundation for cancer gene therapy.This study includes three parts as follows.Part 1 Optimization of Ultrasound-targeted Microbubble Destruction Enhanced GeneTransfection In Vitro Objective 1.To investigate different parameters of ultrasound andtransfectionas well as culture conditions that would affect cell viability,sonoporation andtransfection rate of gene in cancer cells.2.To establish the optimal parameters ofultrasound-mediated microbubble destruction (UTMD)which enhanced gene transfection,3.To evaluate the transfection efficiency and safety of different acoustic microbubbles invitro in order to obtain higher transfection efficiency with minimal effect on cell viabilityand structural integrity of plasmid DNA.4.To evaluate the feasibility of delivery andlocalization of plasmid in subcutaneous transplantation tumors of human cervicalcarcinoma (Hela)in nude mice facilitated by UTMD and to optimize the correlatedparameters in vivo.Methods HeLa cells were cultured in vitro using two different protocolsin two groups,Group A:24 hrs culture for complete adherence;Group S:cell suspension.Subsequently,cells were transfected by different ultrasound intensity,duty cycle andexposure time.Gene transfection (DsRed)and cell viability were evaluated.Usingmicroscope and scanning electron microscopy (SEM),the changes of shape and thesonoporation on cell membrane induced by UTMD were observed.The influence ofparameters on transfection efficiency and cell viability of different cell lines (Hela andIshikawa)using two different DNA plasmids,DsRed and luciferase recombinant plasmid(pCMV-LUC),were studied.The different parameters of UTMD were systematicallystudied to optimize gene transfection,i.e.such as DNA concentration,duty cycle and theduration of exposure.The enhancement of SonoVue microbubble under UTMD conditionwas analyzed.The effects of sonication on plasmid DNA were investigated.Enhancedeffects of liposome microbubble (LM)under UTMD conditions were studied.Differentconcentrations of LM and ultrasound parameters were optimized.Treatment parametersoptimized in HeLa cells were applied in 4 other cell lines (HepG₂,Ishikawa,MCF-7 andB 16-F10)and compared with the transfection of PEI.In transplanted tumors in vivo study,LM and two different plasmids (EGFP and DsRed)were injected by tail vein into the nudemice followed by ultrasound exposure.Duration of gene expression (1～7 d),plasmid usage(20～50μg),localization and safety were evaluated.Results Low intensity and duty cyclehave no great impact on cell viability.Cell injury were found to increase progressively withhigh intensity and duty cycle (P＜0.01),and cell detachment was significantly accompaniedby ultrasound exposure in adherent HeLa cells.Results of factorial design showed that the culture conditions and the ultrasound parameters had interaction between them (P＜0.01).SEM demonstrated that the phenomenon of transient pores in the cell membrane undersuitable ultrasound irradiation was observed in details.The ideal sonoporation conditionsthat cell viability was above 80% and more membrane holes were noted to be at 1.0 W/cm²exposured 3 min with a duty cycle of 20% in cell suspension.The increased transfectionefficiency was related to plasmid concentration and the highest transfection efficiency wasobtained when DNA concentration was 30μg/well.Compared with the ultrasoundirradiation alone,transfection efficiency of optimal UTMD was significantly increased(P＜0.01).The results of agarose gel electrophoresis indicated that the structural integrity ofplasmid DNA was unaffected by the optimal ultrasound parameters.LM and ultrasoundexposure increased transfection efficiency in cultured Hela cells significantly (P＜0.01).Transfection efficiency was the most prominent under the condition of 6% LM.Noapparent cell damage was found in the all groups.These experiments also revealed thatresponses to ultrasound treatment were different for all tested cell lines,dead andtransfected cells in the treated groups were different from the non-irradiated groupssignificantly.In in vivo study,strong fluorescence expression was seen in P+UTMD groupin which gene expression was significantly higher than that in any other groups (P＜0.01).Gene expression level at third day post ultrasound exposure was significantly higher thanany other time points (P＜0.01).There was not significantly expression level between twodifference reporter genes (P=I.000).No tissue damage was seen histologically.Conclusions Ultrasound and transfection parameters,culture conditions have a greatimpact on gene delivery efficiency and cell viability.The optimized parameters andconditions could decrease the cell injury and have a great impact on the sonoporation.Acoustic microbubbles by UTMD could enhance effectively without apparently adverseeffect in vitro and in vivo.UTMD would be an effective,noninvasive gene transfectionmethod and provide a novel,safe non-viral alternative to current gene therapy.Part 2 Study of Ultrasound-Targeted Microbubble Destruction Enhanced GeneDelivery Accompanied with PEIObjective 1.To study the optimized condition of transfection efficiency enhanced by UTMD combined with PEI.2.To observe whether the combination can have asynergistic effect to increase gene transfection.3.To determine whether it could enhancegene delivery in vivo in tumor xenografts.Methods In in vitro study,MCF-7 cells weretransfected with the compounds prepared by the plasmids encoding luciferase(pCMV-LUC)and PEI.SonoVue microbubble was added to the cell suspension or culturedwith PEI/DNA before ultrasound irradiation.The strategy of ultrasound irradiation wasoptimized.The luciferase expression and cell viability were evaluated.Furthermore,theinfluencing factor,such as the plasmid concentration,incubation time,serum,the type ofsolvent and the volume of culture media,were examined.Moreover,two differentmolecular weights (MWs,25 kDa,750 kDa)of PEI were incubated with two differentplasmids (DsRed,pCMV-LUC)to prepare the cationic compounds (PEI/DNA),accordingto the N/P ratio (nitrogen/phosphate ratio).Formation of PEI/DNA complexes wereconfirmed by the gel retardation assay.Cell viability was assessed by MTT Assay.Theeffects of different MWs on their gene expression and the enhancement of ultrasound wereevaluated.In in vivo study,Hela cells were planted subcutaneously in both flanks of femalenude mice.Tumor-bearing mice were administered by tail vein with PBS,plasmid,plasmidand SonoVue microbubble,PEI/DNA and SonoVue microbubble.One tumor was exposedto ultrasound irradiation,while the other served as control.The feasibility of targeteddelivery and tissue specificity facilitated by UTMD and PEI was investigated.Geneexpressions and histology analysis were detected.Results Ultrasound irradiation under anappropriate condition could enhance the gene transfection of PEI/DNA complexes.Incontrast to the PEI/DNA complex alone without ultrasound irradiation or ultrasoundirradiation alone,the combination of UTMD and PEI had a significantly enhancedluciferase activity (P＜0.01).The 2 h pre-irradiation incubation with PEI/DNA complexexhibited a significantly enhanced luciferase activity (P＜0.01).Besides,serum,type ofsolvent and the volume of culture media could affect the transfection efficiency.Electrophoresis experiments revealed that PEI could condense plasmid DNA efficientlywhen the N/P ratio was 3 or higher,and two different plasmids were similar in thiscondition.Cytotoxicity was related to the concentration of PEI.Ultrasound irradiation,evenwithout microbubbles,could significantly enhance luciferase activity of naked plasmid orcationic compound (P＜0.05).The enhancement was significant for the PEI/DNA compound when compared with the naked plasmid (P＜0.05).Gene transfection of 25 kDawas significantly better than 750 kDa (P＜0.01).In in vivo study,the increase of transgeneexpression was related to UTMD with the presence of PEI dramatically.At least 10-foldincrease of luciferase gene transfer was obtained in irradiated tumors compared tonon-irradiated controls (P＜0.01),111-fold increase compared to UTMD alone (P＜0.01).There was not significantly gene expression in other organs or tissues regardless ofultrasound exposure (P＞0.05).No tissue damage was seen histologically.ConclusionsThese results suggest that the combination of UTMD and PEI has a synergistic effect ongene transfection and optimized ultrasound and transfection parameters provide efficientgene delivery in cancer cells.This is a simple and promising method to enhance the geneexpression.Meanwhile,the novel combination can enhance targeted gene delivery andreporter gene expression in tumors at intravenous administration.It is a promising,new andsafe method for gene therapy in vivo.Part 3 Study of UTMD Associated with RNAi Techniques to Silence SurvivinExpression and Induce Cell ApoptosisObjective 1.To construct the recombinant expression vectors targetedSurvivin gene and to analyze the silencing effect by RNAi in cervical cancer (HeLa)cellline.2.To investigate the gene silencing,apoptosis induction and the suppression ofproliferation in vitro and in vivo transfected by UTMD techniques associated with RNAiTechniques.Methods Three survivin-shRNA expression vectors (pSIREN/S1/S2/S3)and anegative control vector was constructed (pSIREN/con)were constructed.The shRNAvectors were added to Hela cells transfected by Lipofectamine or mediated by UTMD.Theoptimal recombinant plasmid was used in the systematic optimization study of UTMD,while plasmid group (P),ultrasound exposure group (US),SonoVue microbubble group (S),plasmid and SonoVue microbubble group (P+S),plasmid and ultrasound exposure group(P+US)were used as controls,respectively.Moreover,the cells were transfected byLipofectamine (P+L).The expressions of Survivin mRNA and protein were detected byRT-PCR and western blot analysis,respectively.Apoptosis index of transfected cells wasquantified by flow cytometry marked with FITC-annexin V/FITC and 7-AAD.In the in vivo study,nude mice were randomly arranged into 3 groups:control group,plasmidinjection and ultrasound (P+US),P+UTMD group.Frozen section,transfection efficiencyand histological examination were evaluated.Protein expressions of Survivin andproliferating cell nuclear antigen (PCNA),Bcl-2,Bax,Caspase-3,Ki-67,nucleostemin(NS),p53 were investigated by immunohistochemistry.Furthermore,microvessel density(MVD)was detected by CD34 protein expressions and apoptotic index (AI)was measuredby TUNEL method.Results Three recombinant plasmids were successfully constructed.Inthe in vitro study,the plasmids were showed to efficiently and specifically inhibit theexpression levels of Survivin gene mRNA and protein except for the pSIREN/con,whilepSIREN/S3 was significantly higher than other recombinant plasmids (P＜0.05).Theapoptosis rate of P+L group [(31.58±3.12)%] was higher than control groups,but remainedlower than P+UTMD group [(43.86±4.44)%].Apoptosis index in P+UTMD group wassignificantly increased as compared with other groups (P＜0.01).There were apparentapoptosis and DNA ladder in P+UTMD and P+L group.After transfected with pSIREN/S3for 48 h,the inhibition ratio of survivin mRNA and protein in P+UTMD group were83.33%±2.73% and 79.67%±3.55%,respectively.The differences were significant whencompared with other groups (P＜0.01).In transplanted tumors experiment,comparing withthose in C and P+US groups,protein expressions of PCNA,Ki-67,Bcl-2,Survivin,NSwere down-regulated markedly,while those of Bax,Caspase-3 and P53 were up-regulatedsignificantly (P＜0.05).MVD decreased significantly,whereas AI increased remarkably(P＜0.05).Conclusions We suggest that Survivin can be regarded as an ideal target foranticancer therapy.UTMD combined with shRNA technique can induce apoptosis andinhibit proliferation significantly,without causing any apparently adverse effect,representing a new,promising technology that can be used in the tumor gene therapy andresearch.