Sonoporation using microbubbles promotes lipofectamine-mediated siRNA transduction to rat retina

Ultrasound-targeted microbubble destruction(UTMD) has been utilized to deliver naked siRNA into cells in in vitro settings. But whether UTMD can safely deliver naked siRNA into in vivo cells have remained unknown. Th is work was performed to investigate the feasibility of UTMD-enhanced naked siRNA transduction (or combined with Lipofectamine ) in vivo retinal cells and compare the performance between UTMD and ultrasonic irradiation alone in this enhancing eff ect. A dose of Cy-labeled siRNA was injected into the vitreous cavity of rat eyes under the diff erent conditions of Lipofectamine  or/and UTMD. Transduction effi ciency was assessed by fl uorescence microscopy and fl ow cytometry. Cell and tissue damage was assessed by trypan blue exclusion test and hematoxylineosin staining, respectively. Th e quantity and the density of transducted cells in the group received Lipofectamine  and UTMD was far more than that in other groups. Th e number of transducted cells in the group received Lipofectamine  and ultrasonic irradiation alone was slightly more than that in the group received Lipofectamine . Cy-siRNA-positive cells can also seen in the group received UTMD alone, although the transduction effi ciency is extremely low. Cell viability in each group was more than , and retinal architecture in each group was well preserved. Th ese results indicated that UTMD, with a signifi cantly higher performance than ultrasonic irradiation alone, can eff ectively enhance the Lipofectamine -mediated naked siRNA transduction in vivo reinal cells without any cell or tissue damage. Th is method can serve as a novel approach to treat the diseases of eye ground. ©  Association of Basic Medical Sciences of FBIH. All rights reserved


INTRODUCTION
RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) is a powerful gene technology allowing the silencing of mammalian genes with great specificity and potency, which has been widely utilized to down-regulate sequence-specific gene expression for the treatments of various diseases [-].But siRNAs do not readily penetrate the cell membrane.Moreover, they are easily degradable when exposing to nuclease in vivo [-].Therefore, clinical applications of siRNA largely depend on the development of delivery systems that can bring intact siRNA into the cytoplasm of the target cells.For delivery and prolonged expression in vivo, the siR-NA genes are usually constructed on vectors, such as adenovirus, recombinant adeno-associated virus, lentivirus and plasmid [-].This is costly and time-consuming.In terms of the virus vectors, they often bring about some issues about safety due to their pathogenic nature [, ].Although naked plasmids are free of virus-associated adverse effects, their transduction efficiency is low and their transgene expression is relatively poor [].Commercially available cationic lipid formulations, such as Lipofectamine (Invitrogen), RNAifect (Qiagen), have been investigated as potential enhancers of siRNA delivery in vitro.Although they are also eff ective when delivered systemically, cationic lipid-mediated cellular toxicity, elicited inadvertent gene expression and enhanced immune response to siRNA maybe become the obstacles to their widespread use clinically [].Better methods for siRNA delivery are needed.Ultrasound-targeted microbubble destruction (UTMD) is a potential site-specifi c gene transfer modality that has been developed over the past two decades.Recent studies confi rmed that UTMD can eff ectively deliver naked siRNA into B-cell lymphoma, endothelial cells and mesenchymal stem cells in in vitro settings [, , ].But to our knowledge, whether UTMD can safely deliver naked siRNA into in vivo cells have remained unknown.Moreover, our in vitro study confi rmed that UTMD alone can not deliver naked siRNA into human and rat retinal pigment epithelial cells, and it is ultrasonic irradiation (not UTMD) that significantly enhanced Lipofectamine -mediated naked siRNA transduction [].
In the present study, we injected naked siRNA into the vitreous cavity of Wistar rat eyes under the different condi- we hoped to answer the following questions as: ) whether UTMD can effectively enhance naked siRNA or Lipofectamine -mediated naked siRNA transduction to in vivo cells?) which one indeed enhances the Lipofectamine -mediated naked siRNA transduction to in vivo retinal cells between ultrasonic irradiation alone and UTMD ?

Cy-labeled siRNA preparation
Cy-labeled siRNA(Cy-siRNA) was purchased from Ri-boBio Co.,Ltd (Guangzhou, China) and was used to determine transduction efficiency of siRNA, optimize transfection condition and serve as siRNA localization.This RNAi negative control does not have homology with mammal gene and has very good pH tolerance, thus is stable in the living cell, which can be detected by flow cytometry, fluorescence microscope and laser co-focus microscope.Cy labeled spot was on the ' end and this modifi cation didn't infl uence the silence activity of siRNA.A dose of  nmol Cy-siRNA powder was dissolved in  μl diethylpyrocarbonate (Sigma, USA) treated water, then μL Cy-siRNA (. nmol) was drawn out and mixed with μL Lipofectamine (L) (invitrogen, USA).The mixed solution was standing for  minutes before intravitreal injection, ensuring valid transduction efficiency.All the operation process had been away from light.

Microbubble contrast agents
SonoVue® microbubble contrast agent (Bracco, Milan, Italy), a composition of a core of sulfur hexafluoride (SF) gas and an envelope of phospholipids, was reconstituted in saline solution according to the manufacturer's protocol, and yielded a preparation containing -×  microbubbles (MBs)/mL by inversion/agitation of the unit.The average diameter of the MBs was .-.μm.

Animal preparation and grouping
After obtaining the approval of the local ethics committee,  normal adult Wistar rats (male or female, age=- weeks, weight=-g, SLACCAS, Shanghai, China) were enrolled in this experiment.All animals were bred, maintained, and sacrificed humanely in strict compliance with the policies stated in the statement of Association for Research in Vision and Ophthalmology for the use of animals in ophthalmic and vision research.According to the content of intravitreal injection, Wistar rats were divided into six groups as follows (n=): Group  (control): μL sterile pyrogen-free normal saline (NS); Group  (US): μL Cy-siRNA (. nmol), μL NS and an ultrasound exposure; Group  (US+MBs): μL Cy-siRNA (. nmol), μL MBs, μLNS and an ultrasound exposure; Group  (L): μL mixed solution of Cy-siRNA and Lipo-fectamine and μL NS; Group  (L+US):μL mixed solution of Cy-siRNA and Lipo-fectamine, μL NS and an ultrasound exposure; Group  (L+US+MBs): μL mixed solution of Cy-siRNA and Lipofectamine, μL MBs and an ultrasound exposure.

Intravitreal injection
Th e method of intravitreal injection was previously described by Zheng [,].Briefl y, Wistar rats were anesthetized by intraperitoneal injection of  chloral hydrate (mg/kg body weight).Th e pupils were dilated with one drop of  atropine sulfate and tropicamide, and the eyes were gently protruded using a rubber circle and subsequently covered with . ofl oxacin eye ointment (Xingqi, Shenyang, China) to simulate a preset lens.Under a surgical microscope (SM-J, Eder, Shanghai, China), NS, Cy-siRNA or a mixed solution of Cy-siRNA and Lipofectamine , with or without MBs, were injected into the left eye according the grouping using a blunt -gauge Hamilton syringe.Th e right eyes served as a control eye, and were injected with μL NS.

Utrasound exposure
A therapeutic ultrasound machine (Topteam, Chattanooga, TN, USA) and a -cm  probe were applied in this study.Th e parameters of US exposure were as follows: frequency, MHz, power, W/cm  , duty cycle, , pulse recurrent frequency, Hz, duration,  seconds.Immediately after intravitreal injection, a -cm  US probe placed directly onto the conjunctival surface after a small amount of coupling medium was smeared on its face, then the insonation was performed.

Retina-stretched preparation and fl uorescence imaging
Six eyes of each group were harvested at  hour after intravitreal injection.Fundus oculi were prepared after enucleation of the globe by removing the anterior segment with a blade and carefully transferring the whole retina to a microscope slide.Six relieving incisions were made to allow the retina to be fl attened.Immediately, the quantity and density of Cy-siRNA-positive cells in retina were observed and photographed by an inverted fluorescent stereoscope (ZEISS, Stemi SV, Jena, German).Th e area and mean grey of Cy-siRNA fluorescence were analyzed and quantified using Axiovision . software (Carl Zeiss, Goettingen, Germany).Data were presented as integrity optical density (total area × mean grey of Cy-siRNA).

Flow cytometry and cell viability
Single-cell suspensions were prepared from six eyes of each group at  hour after intravitreal injection as previously described by Portillo [].Briefly, retinas were isolated and minced following by digestion in a solution containing  IU/ml papain and  μg/ml DNase (Worthington Biochemicals, Freehold, NJ) for  min at  °C.Tissue was dissociated by gentle pipetting and passed through a  μm cell strainer.Flow-through was mixed with Fetal bovine serum (FBS; HyClone Laboratories Inc.South Logan, UT) and washed.Tissue trapped by the strainer was digested with  mg/ml collagenase type I (Worthington Biochemicals) for  min at  °C to free endothelial cells.After dissociation and mixing with FBS, cells were washed once in Dulbecco's modifi ed Eagle's medium(DMEM;Gibco, Grand Island, USA) with  FBS for  min at  g at room temperature.Cells obtained after papain-DNase and collagenase treatments were pooled.Finally, Cy expression of the infected cells was quantitatively examined by flow cytometry (EPICS XL, Beckman Coulter Co, Miami, USA) analysis.Data were presented as Cy-siRNA-positive cell ratio (, the number of infected cells per  retinal cells).In addition, cell viability was assessed by trypan blue exclusion test.Cells were suspended in PBS.Ten μL of cell suspension were mixed with an equal amount of . trypan blue dye (Invitrogen, USA).Blue (dead) and white (living) cells were counted microscopically in a hemocytometer (Sigma, USA).

Histopathologic examination
Two eyes of each group were harvested at  hour after intravitreal injection.The eyes were enucleated, and fixed in  formaldehyde solution at a room temperature.Thereafter, they were embedded in paraffin, and cut into μm-thick sections.Subsequently, the sections were stained with hematoxylin-eosin(HE) to observe retinal architecture, infl ammatory cell infi ltration, and proliferative membrane using light microscopy (Zeiss Axiovert S , Jena, Germany).All the results of histopathologic examination were confirmed by two masked expert pathologists.

Statistical analysis
Data were expressed as the means and standard deviation (mean±SD).Analysis of variance (ANOVA) was used to determine the significance of the difference in a multiple comparison.Differences were considered significant at p<..All statistical analysis was performed with SPSS version  software for Windows (SPSS Inc, Chicago, IL).

L L+US L+US+MBs
The number of transducted cells in Group (L+US) was slightly more than that in Group  (L).In addition, the quantity of Cy-siRNA-positive cells in Group  (L) is signifi cantly more than that in Group  (US+MBs).As for Group  (US) and Group  (control), none of the fl uorescence can be found.
Transduction effi ciency of Cy-siRNA in retinal cells under diff erent conditions is shown in Figure .Th e ratio of Cy-siRNA-positive cells in Group  (L+US+MBs) is the highest (.±.),which is far more than that in other five groups (L+US, L, US+MBs, US, control).From Group  to Group , the ratios of Cy-siRNA-positive cells decreased in order (.

L L+US L+US+MBs
the cell membrane [,], and hence an increased uptake of siRNA.In the sonification zone, cavitation also creates small shock waves that increase cell permeability by disruption of the membrane barrier [].Ultrasound irradiation triggers two liposomal siRNA-release mechanisms: the predominant one is diffusion through liposome membrane, and the less signifi cant one is liposome disintegration [].
Compared to our in vitro study [], this in vivo study produce different results as mentioned above.Although the results could be simply explained by varied degrees of physical impacts, the subtle mechanisms underlying these phenomena require further study.In addition, the transduction effi ciency of siRNA in this in vivo study is relatively low, which may result from the diff erent characteristics of contrast agents (shell gas properties and parcels), the different cells and the diff erent target genes used in this study.
As one of conventional diagnostic techniques of clinical imaging, the safety of ultrasound has been established.But the investigation about the safety of UTMD in the therapy of ophthalmological diseases has not yet been fully fi nished.In our study, we referred to the condition previously described by Zheng [,]: frequency, MHz; power, W/cm  ; duty cycle,; pulse recurrent frequency,Hz; duration, seconds; MBs concentration, .Because this UTMD condition have no obvious tissue damage to the retina assessed by histopathologic examination.In the present study, cell viability in each group was more than , and retinal architecture in each group was well preserved, which indicated that this dose of Lipofectamine (. nmol) and this UTMD condition mentioned above have no signifi cant adverse eff ects on retinal cell viability and retinal architecture.

CONCLUSION
Th is study demonstrated UTMD can eff ectively enhance the Lipofectamine -mediated naked siRNA transduction to in vivo reinal cells, which reduces the dose of liposome and the attendant adverse effects.Although some limitations present, UTMD-enhanced liposome-mediated siRNA transduction to retina can serve as a novel approach to treat the diseases of eye ground.Further studies are needed to evolve so that an optimal condition of UTMD and an appropriate dose of liposome can be obtained.Meanwhile, new types of microbubble contrast agents require further study.
BOSNIAN JOURNAL OF BASIC MEDICAL SCIENCES 2011; 11 (3): 148-152 XIAOZHI ZHENG ET AL.: SONOPORATION USING MICROBUBBLES PROMOTES LIPOFECTAMINE MEDIATED SIRNA TRANSDUCTION TO RAT RETINA tions of Lipofectamine  or/and UTMD.By the quantifi cation of transduction effi ciency of naked siRNAs in retina, -siRNA-positive cells in retina-stretched preparation are shown in Figure  and Figure .Th e quantity of transducted cells in Group  (L+US+MBs) was far more than that in other five groups (L+US, L, US+MBs, US, control), and the density of Cy-siRNA -positive cells in this group is the highest.

DISCUSSION
Th e results presented here indicate that UTMD can eff ectively enhance the Lipofectamine -mediated naked siRNA transduction to in vivo reinal cells without any cell or tissue damage.It is UTMD that indeed enhances Lipofectamine -mediated naked siRNA transduction to in vivo retinal cells, with a signifi cantly higher performance than ultrasonic irradiation alone in this enhancing effect.UTMD alone can also slightly enhance naked siRNA transduction to in vivo cells, although the transduction effi ciency is extremely low.The detailed mechanism of UTMD-enhanced naked or liposome-mediated siRNA transduction has not yet been fully explained.It is considered that bioeff ects of UTMD, such as cavitation, thermal effect, radiation force, and chemical effect together may result in permeability changes of
 ± ., . ± ., . ± .), and there is no Cy-siRNA expression in the retinal cells of Group  (US) and Group (control) detected by flow cytometry.Th e eff ects of Lipofectamine,US and US plus MBs on cell viability assessed by trypan blue exclusion test are shown in Figure .Th e retinal cell viability  hours after transfection in Group (control),Group  (US) and Group  (US+MBs) was . ± ., . ± ., and . ±., respectively, and it was . ± ., . ± . and . ± . in Group  (L), Group (L+US) and Group  (L+US+MBs), respectively.Histological observation of each group using HE-staining of retinal architecture in Wistar rats at  hour after intravitreal injection is shown in Figure .Apparently, all layers of the retina were well preserved without photoreceptor loss, nuclear layer vacuolation, or infl ammation under this condition.