Inhibition Of Hepatic Fibrosis With Artificial MicrorRNA Using Ultrasound And Cationic Liposome-bearing Microbubbles

Backgrounds and objectivesHepatic fibrosis is a common pathological change in chronic liver injury characterisedby the accumulation of extracellular matrix (ECM). Following liver injury, hepatic stellatecells (HSCs) become activated and transform into myofibroblast-like cells that expressalpha-smooth muscle actin (α-SMA) and produce ECM.Transforming growth factor β1(TGF-β1) has been considered one of the most potent fibrogenic mediators to fibrosis.Because of its multiple actions, complete blockage of TGF-β1may have serious side effects.Connective tissue growth factor (CTGF) has received significant attention as a downstreameffector of the TGF-β1fibrogenic effect. CTGF not only can lead to the activation,proliferation and migration of primary HSCs, but can also promote the accumulation of ECMcomponents and inhibit their degradation. Disruption of CTGF expression has been shown toeffectively suppress the activation of HSCs and the accumulation of ECM. CTGF expressionis low or absent in normal liver tissue; however, its expression progressively increases infibrotic liver. CTGF expression is significantly correlated with the development of hepaticfibrosis. Strategies to block CTGF by small interfering RNAs (siRNA) have achievedfavourable anti-fibrotic effects for treating hepatic fibrosis.Based on recent studies, microRNAs (miRNAs) have gained significant attention for thetreatment of hepatic fibrosis. Artificial microRNAs (amiRNAs) exploit the backbone ofnatural miRNAs to generate designed miRNAs that can efficiently silence the gene of interest.This strategy has produced highly efficient and specific gene silencing for therapeuticapplications. However, the challenge is to effectively deliver amiRNAs to the target organ.Ultrasound-targeted microbubble destruction (UTMD) has emerged as a novel non-viralgene delivery technique because of its safety, high efficiency and local gene transfer. This method involves the attachment of genes to microbubbles, which are then injected andcirculated through blood vessels and destroyed at the target site by ultrasound insonation. Thedestruction increases capillary permeability and generates transient holes in the cellmembrane and releases the payload, which is incorporated intracellularly. Microbubbles,which consist of a lipid shell encapsulating perfluorocarbon gas. Nevertheless, thegene-loading capacity of microbubbles remains a consideration. Fortunately, studies haveshown that the loading capacity can be increased by attaching cationic liposomes to the lipidshell of microbubbles.In this study, we utilised novel cationic liposome-bearing microbubbles combined withultrasound to transfect a plasmid-based amiRNA designed against CTGF mRNA and protein,which showed high gene-silencing efficacy in vitro, to determine whether this techniquecould efficiently suppress the expression of CTGF and exert antifibrogenic effects on hepaticfibrosis in vivo.Methods1. To construct microRNA eukaryotic expression vectorsFour different sequences targeting CTGF genes were designed. And also designed ascramble control sequence.The modified pre-miRNA sequence structure was derived frommurine miR-155. These sequences were annealed and ligated into five pcDNA6.2-GW/EmGFP-miR plasmids. Recombinant plasmid named miRNA-CTGF-1, miRNA-CTGF-2,miRNA-CTGF-3, miRNA-CTGF-4and miRNA-CTGF-Scramble.Used liposome carrying method to transfect five recombinant plasmids into rat hepaticstellate cells(HSC-T6) respectively and the blank contrast only transfect the Lipofectamine2000. After24hours, observed the EmGFP expression in HSC-T6cells through invertedfluorescence microscope and evaluated there transfection efficiency.Total RNA was extractedafter incubation for48h. The protein was extracted after incubation for72h. Quantitativepolymerase chain reaction (qPCR) and western blot analysis detected the differenceexpression level of CTGF mRNA and protein in HSC-T6cells, testified to the specificityinhibitional effect of miRNA-CTGF to target genes, and specified that which recombinantplasmid had the strongest inhibitional effect and its miRNA sequence.2. To investigate the effects of diagnostic ultrasound targeted microbubble destructionon permeability of normal liver in rats as well as its hepatic and renal toxicity. One hundred and four rats were divided into four groups, including the group ofultrasound irradiation with frequency of1.5/3.2MHZ combined with microbubble, the groupof ultrasound irradiation only, the group of microbubble only and the control group. Thepermeability of capillary and cell membrane was detected by using Evans blue and lanthanumnitrate as tracers, respectively. Its effect on normal rat liver tissue permeability wasobserved by confocal laser microscope. Blood chemical analysed the serum ALT,AST,BUN and CREA levels.3. To prepare DNA-loading cationic liposomes bearing-microbubbles.Biotinylated ultrasound microbubbles and biotinylated cationic nanoliposomes wereprepared by cryochem and machine vibration．Physicochemical properties were detected.Toobtain DNA-loading liposomes, incubated the biotinylated cationic nanoliposomes and DNAplasmid at room temperature for30min. Gel retardation assay for determining the mass ratiofor the biotinylated cationic nanoliposomes complete complexation with DNA. The newDNA-loading microbubbles were made by combined microbubbles with DNA-loadingcationic nanoliposomes by biotin-avidin system. And the morphology and connect effect ofcomplex were observed by laser scanning confocal microscope. The loaded-gene ability ofthe new microbubbles were detected.4. Inhibition of hepatic fibrosis by Artificial microRNA using cationic liposome-bearingand ultrasoundThe rats were randomly divided into eight groups: the normal control group (n=8),dimethylnitrosamine (DMN, Sigma, St. Louis, MO)-induced model group (n=8), theamiScramble group (amiScramble plasmid delivered via gene-loaded cationic microbubblecombined with ultrasound, n=8) and the amiCTGF groups (n=40). The amiCTGF groups weredivided into five groups randomly (eight rats per group): amiCTGF+M++US group(amiCTGF plasmid delivered via gene-loaded microbubble combined with ultrasound),amiCTGF+M+US (amiCTGF plasmid delivered via common microbubble combined withultrasound), amiCTGF+M+group(amiCTGF plasmid delivered via gene-loaded microbubble),amiCTGF+M group(amiCTGF plasmid delivered via common microbubble) and plasmidgroup(amiCTGF plasmid only).In the normal control group, rats received intraperitoneal saline injections for threeconsecutive days per week for up to4w. In the DMN model group, amiScramble group and amiCTGF groups, rats received intraperitoneal1%DMN (1ml/kg body weight) injections forthree consecutive days per week for up to4w. At the end of the second and third weeks, theDMN model group received a slow bolus injection of0.5ml/kg body weight of microbubbles,followed by1ml of saline to wash the tube via the caudal vein and ultrasound was applied asdescribed. At the end of the second and third weeks, the amiScrambe group and the amiCTGFgroups were given according treatments with amiScramble plasmid and amiCTGF plasmidrespectively.All rats were sacrificed4w after DMN or saline administration under generalanaesthesia. The liver tissue was quickly removed and a portion was instantly frozen in liquidnitrogen for Western blot and real-time RT-PCR analysis. Another portion was fixed in10%phosphate-buffered formalin for histopathological studies.Results1. Successfully constructed four MicroRNA plasmid expression vectors ofCTGFmRNAsequences，consequence of sequence analysis of those plasmids was coincidedwith computer designs. Green fluorescence could be observed in post-transfectional HSC-T6cells, which convinced that cell transfections were successful, and transfected plasmidsexpressed in cells. qPCR and Western blot showed that there was no obviously decline inCTGF mRNA and protein expression in the scambled amiRNA group(amiScamble) ascompared with the untreated HSC-T6cells group(blank) and that the four artificial microRNAtargeting CTGF groups showed different degrees of inhibitory effect, of which miRNA-CTGF-4group exhibited the strongest inhibitory effect.2. Evans blue amount in group of ultrasound combined with microbubble was highersignificantly than that in the other three groups (P<0.01).Lanthanum nitrate-tracingtransmission electron microscope examination indicated that intracellular lanthanum could befound entering the hepatocytes in group of ultrasound combined with microbubble. Bloodchemical analysis indicated the serum ALT, AST levels was increased (P <0.01) in group ofultrasound combined with microbubble at0.5h and12h when compared to the other threegroups, but there was no significant difference at24h (P>0.01). There was no significantdifference in the serum BUN and CREA levels among the four groups after treatment. Therewas cellular swelling in liver cells in group of ultrasound combined with microbubble at0.5h,but it repaired after one week. 3. The biotinylated cationic liposomes appeared as spherical or nearly sphericalnanoparticles. The mean zeta potential was50mV. A gel retardation assay showed that thebiotinylated cationic liposomes/DNA volume ratio required to completely retain DNA in thecomplex was6:1．It showed that the green wall of biotinylated microbubbles was surroundedwith a number of red small round DNA-loading nanoliposomes. Its gene loading ability wasmuch higher than common microbubble. Contrast imaging showed that gene-loaded cationicmicrobubble could significantly enhance echo intensity of rat liver.4. The amiCTGF treatments prevented the development of hepatic fibrosis induced byDMN, as confirmed by HE, Masson’s trichrome, and Sirius red staining, of which amiCTGFdelivered via gene-loaded cationic microbubble combined with ultrasound exhibited thestrongest inhibitory effect. The expression levels of CTGF mRNA and proteins were very lowin the normal control group. But they were significantly increased in the model andamiScrambe treated groups compared with that in the normal control group (P<0.01), ofwhich amiCTGF delivered via gene-loaded cationic microbubble combined with ultrasoundexhibited the strongest inhibitory effect. However, they were markedly decreased afteramiCTGF treatment compared with those of the model and the amiScrambe treatedgroups.Moreover, compared with the model and amiScrambe treated groups, TGF-β1andα-SMA protein levels was significantly reduced after amiCTGF treatment in terms of bothquantity and intensity as determined by immunohischemistry, of which amiCTGF deliveredvia gene-loaded cationic microbubble combined with ultrasound exhibited the strongestinhibitory effect.Conclusions1. Successfully constructed four microRNA plasmid expression vectors of CTGFmRNA sequences; all of them inhibit CTGF mRNA and protein expression and the inhibitionrate of miRNA-CTGF-4is the most.2. diagnostic ultrasound targeted microbubble destruction can increase the capillary andcell membrane permeability of normal liver without significant increase in hepatic and renaltoxicity.3. A new high gene-loading microbubble was developed by conjugating gene-loadingliposomes and microbubbles, using the high affinity interaction between avidin and biotin,which had good gene carrying capacity without changing its acoustic properties．4.Gene silencing of CTGF using amiRNAs delivered via liposome-bearing microbubbles combined with ultrasound may be a valuable specific approach for prevent hepaticfibrogenesis.