rna interference ( rnai ) is a conserved cellular mechanism by which a small double stranded rna ( dsrna ) directs the degradation of complementary mrna and therefore inhibits the expression of a specific gene.(1 ) since its discovery , rnai has become a powerful tool to study gene functions in biological processes . the ability to induce rnai in mammalian cells using synthetic small interfering rna ( sirna ) has stimulated great interest in therapeutic applications of rnai . in numerous studies , sirnas have shown promise for treating a variety of diseases , including influenza and hiv infection , cancer and genetic defects . a key challenge of rnai - based therapeutic application is the efficient delivery of sirna into target cells . sirna is usually 21 nucleotides in length and highly charged and therefore can not cross the cytoplasmic membrane by free diffusion . in the circulation and interstitial space , sirna is vulnerable to degradation by rnase.(11 ) although sirna can be delivered directly and locally to the target sites in limited applications , a carrier system is required in most applications to protect sirna from degradation and to facilitate its uptake by target cells . the existing carrier systems usually contain a key cationic component , such as a cationic lipid , a cationic polymer or a cationic peptide , in order to bind sirna effectively . other supplementary components help to improve the stability , solubility or pharmacological profiles of sirnacarrier complexes.(16 ) the use of carriers can dramatically increase the uptake of sirna . however , the efficacy of the carriers in mediating functional sirna delivery , i.e. , leading to the knockdown of the target transcript , remains low.(17 ) many of the existing carriers were originally designed for dna delivery , and hence have not been optimized to account for the differences between sirna and dna . sirna mediates its effect in the cytosol,(18 ) while dna requires entry into the nucleus in order to gain access to the transcriptional machinery . such differences between sirna and dna could have great influences on the functionality and efficacy of a carrier . to rationally develop efficient sirna carriers , it is necessary to elucidate the mechanisms by which the carriers mediate functional sirna delivery . however , the current knowledge on the mechanisms of the carriers comes predominantly from studies with dna delivery . although different carriers have vastly different chemical structures and physical properties , it is believed that a majority of the dnacarrier complexes is taken up into the cells by endocytosis . a primary focus in the research and development of carriers for dna delivery has been to facilitate the endosomal escape of the dnacarrier complexes prior to their degradation in the lysosomes . however , even with the most efficient dna carriers developed so far , it remains controversial to what extent the escape from endosome contributes to transfection efficiency . considering the physical differences between sirna and dna and their sites of action , whether endosomal escape plays any significant role in functional sirna delivery by the various carrier systems has yet to be determined . in this report , we have determined the mechanisms by which a widely used cationic lipid carrier mediates functional delivery of sirna by a combination of imaging , pharmacological and genetic approaches . we show that although most of the cellular uptake of sirna lipoplexes is via endocytic pathways , these pathways do not appear to play a significant role in the delivery of sirnas that mediate rnai . instead , a novel pathway , probably mediated by direct fusion between sirna lipoplexes and the plasma membrane , is responsible for the functional delivery of sirna into the cell . african green monkey kidney epithelial cells bsc-40 , human embryonic kidney cells 293 ft , and human cervical carcinoma cells hela were purchased from the american type culture collection ( atcc ) . the cells were maintained in dulbecco s modified eagle s medium ( dmem ) , supplemented with 100 iu / ml of penicillin , 100 iu / ml of streptomycin , 2 mm l - glutamine , and 10% heat - inactivated fetal bovine serum ( fbs ) ( biowest , miami , fl ) . the cells were cultured in 6-well falcon tissue culture plates in a 5% co2 humidified incubator at 37 c . sirnas specific for cyclophilin b ( cyb ) and green fluorescent protein ( gfp ) and cy5 conjugated gfp sirna were purchased from dharmacon ( lafayette , co ) . the sirna sequences are as follows : gfp sirna : ( sense ) 5-ggcuacguccaggagcgcadtdt-3 and ( antisense ) 5-ugcgcuccuggacguagccdtdt-3. cyb sirna : ( sense ) 5-ggaaagacuguuccaaaaadtdt-3 and ( antisense ) 5-uuuuuggaacagucuuuccdtdt-3. the wild - type and mutant dynamin expression plasmids were generously provided by dr . mark mcniven of the mayo cancer center(25 ) and were cloned into pegfp - n1 expression vector from clontech . the vectors expressing the wild - type and mutant cav - fed and cav - dn caveolins were generously provided by dr . jan eggermont(26 ) and were cloned into the pcineo / ires - gfp expression vector . mutant cell lines were established by electroporation of bcs cells followed by selection in 2.5 mg / ml geneticin ( invitrogen , carlsbad , ca ) . the day prior to experiments , the cells were sorted with a facsaria ( becton - dickinson and co. ) to enrich for the gfp positive cells . uptake of fluorescence - labeled transferrin ( sigma - aldrich ) was used to test if the k44a mutant cells lacked the clathrin - mediated endocytosis . uptake of fluorescence - labeled cholera toxin ( invitrogen ) was used to test if the caveolin mutant cells lacked the caveolin - mediated endocytosis . sirna lipoplexes were prepared following the manufacturer s protocol . in brief , 4 l of dharmafect1 was diluted into 200 l of opti - mem i ( invitrogen ) and then mixed with an equal volume of opti - mem i with appropriate amount of sirna . the sirna lipoplex solution was then diluted 5-fold in dmem , 10% fbs for transfection . twelve hours prior to transfection , bsc cells were seeded in 6-well plates at a density of 2  10 cells / well so that , by the time of transfection , the cells were in the early log phase and reached 70% confluence . during transfection , cell culture media were aspirated and cells were washed with pbs . unless otherwise specified , 2 ml of 100 nm sirna lipoplexes was incubated with cells for 4 h at 37 c . the cells were subsequently washed using cellscrub buffer ( genlantis , san diego , ca ) and pbs . after washing , fresh media were added and cells were cultured at 37 c for the indicated length of time . pharmacological inhibitors were purchased from sigma - aldrich ( st . louis , mo ) and were used at the following concentrations : cytochalasin d , 100 m ; chlorpromazine , 10 g / ml ; filipin , 10 g / ml ; nystatin , 100 g / ml ; amiloride , 3 mm ; d , l - threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol ( pdmp ) , 10 m ; sodium azide , 40 mm ; and 2-deoxy glucose , 40 mm . the cells were pretreated with the inhibitors for 1 h at 37 c and then transfected with sirna lipoplexes for 4 h at 37 c in the presence of the inhibitors . the cells were washed and cultured in fresh media in the absence of the inhibitors for another 20 h prior to rna isolation . in experiments where sirna transfection was carried out at 4 , 10 , 20 , and 37 c , the cells were incubated at specified temperatures for 1 h and then transfected with sirna lipoplex at the same temperatures for 4 h. the cells were washed , replenished with fresh media , and incubated for 20 h at 37 c prior to rna isolation . at indicated hours post - transfection , single cell suspension was prepared and total rna was extracted using an rneasy mini kit ( qiagen , valencia , ca ) according to the manufacturer s protocol . rna concentration was determined by uv absorbance at 260 nm , and all samples had an a260/a280 ratio greater than 1.95 . total rna was first converted into cdna using random hexamers and taqman reverse transcription reagents ( applied biosystems , foster city , ca ) . the levels of cyb and actin mrnas were then measured using taqman gene expression assays ( applied biosystems ) . the real time rt - pcr reactions were performed in applied biosystems microamp optical 96-well reaction plate , on a prism 7000 real - time pcr system . unless specifically indicated , 2 ml of 100 nm sirna lipoplexes were incubated with cells for 4 h at 37 c . the cells were washed with cellscrub buffer , trypsinized and suspended in pbs plus 2% bovine serum albumin and 0.05% nan3 . the cells were analyzed with a facscaliber and cellquest software ( becton - dickinson ) . cellular uptake of cy5 labeled sirna was quantified by measuring the relative fluorescence signal in cy5 channel . cells incubated with nonlabeled sirna lipoplexes were used as a control , and their mean fluorescence values were subtracted from the uptake measurements of cy5-sirna lipoplexes . cells were grown in 35 mm diameter mattek ( mattek , ashland , ma ) glass bottom culture dishes , transfected with cy5-sirna lipoplexes , and viewed on an inverted epifluorescence microscope ( olympus ix71 , olympus , center valley , pa ) , integrated in a deltavision deconvolution system ( applied precision , issaquah , wa ) . cells were housed in an environmental chamber ( solent scientific , segensworth , u.k . ) to maintain constant temperature , humidity and co2 levels during the observation . unless specified , all experiments were conducted at 37 c , using an atmosphere of 95% air and 5% co2 . for fluorescence imaging , cell nuclei and endolysosomes cells were viewed in opti - mem i medium with an olympus 63 planapo oil immersion objective with a numerical aperture of 1.40 , fitted with a coolsnap hq monochrome 12-bit cooled ccd camera ( photometrics , tucson , az ) . images of 512  512 pixels were captured by applied precision s softworx software and analyzed using imaris 4.2 ( bitplane , saint paul , mn ) . to elucidate the mechanisms underlying the functional sirna delivery , we focused on dharmafect1 , a widely used sirna transfection reagent based on cationic lipids . for easy imaging analysis , we used african green monkey kidney epithelial cells bsc-40 . human embryonic kidney cells 293 ft and human cervical carcinoma cells hela were also used in certain experiments . to measure the functional sirna delivery , we assayed the knockdown of an endogenous gene cyclophilin b ( cyb ) because it is ubiquitously expressed and its knockdown has no apparent effect on cell survival and growth . cyb is also highly conserved between human and african green monkey ; therefore , the same sirnas are effective in cells derived from both species . we used quantitative pcr to measure the level of cyb transcript so as to detect the effect of functional sirna delivery as directly and quickly as possible . in addition , we established that the uptake of sirna lipoplexes is proportional to sirna concentration in a range of 10 pm to 200 nm , and the target transcript knockdown is linear to sirna concentration in a range of 100 pm to 100 nm ( supplementary figure s1 in the supporting information ) . because 100 nm sirna produces the most dramatic knockdown of the target transcript but is still within the linear range , we used 100 nm sirna in most experiments . to determine the dynamics of sirna lipoplex transfection , we analyzed the localization of sirna lipoplexes inside the cells at different times post - transfection . bsc cells were incubated with cy5 labeled sirna lipoplexes for 1 , 2 , or 4 h , washed and then fixed for imaging analysis . some of the 4 h transfected cells were washed and cultured for an additional 4 , 12 , and 46 h before fixation and imaging analysis . sirna lipoplexes were detected in cells following 1 h transfection ; the number of sirna lipoplexes increased significantly following 4 h transfection ( figure 1a ) . a significant number of sirna lipoplexes still persisted inside the cells 46 h post - transfection . cy5-sirna lipoplexes were incubated with bsc cells for the indicated length of time , washed , and cultured for the indicated length of time before fixation and imaging analysis . bsc cells were transfected with cy5-sirna lipoplexes for the indicated length of time in the presence of lysosensor . the number of intracellular sirna complexes and the number of sirna complexes that overlapped with lysosensor staining were enumerated . percentages of intracellular sirna complexes that colocalize with lysosensor are shown at the indicated time points . bsc cells were transfected with cyb - specific sirna lipoplexes for 2 or 4 h , washed , were used for rna isolation immediately . some 4 h transfected cells were cultured for another 2 or 20 h before rna isolation . the levels of cyb transcript were quantified by real - time pcr and normalized to that in nontransfected cells . thus , cells were stained with lysosensor specific for endolysosomes and then transfected with sirna lipoplexes for 1 , 2 , 3 , and 4 h. quantification of colocalization of lysosensor and cy5-labeled sirna lipoplexes revealed that > 90% sirna lipoplexes were localized in the endolysosomes ( figure 1b ) . to determine the relationship between the observed uptake of sirna lipoplexes and the target transcript knockdown , bsc cells were transfected for either 2 or 4 h and assayed for cyb transcript level immediately , or transfected for 4 h , washed and cultured for additional 2 or 20 h before measuring the level of cyb transcript . a decrease in cyb mrna level was detected following 2 h transfection , but a significant decrease was clearly evident following 4 h transfection ( figure 1c ) . additional culture of 4 h transfected cells resulted in further decrease of cyb transcript level . by comparing figures 1a , 1b and 1c , it is clear that significant target mrna knockdown occurred in the absence of significant change in the level of sirna lipoplexes in the endolysomes . because a 4 h sirna lipoplex transfection followed by a 20 h culture results in a consistent cyb mrna knockdown by 8090% , we used this assay format in the rest of the study . one of the best characterized and most important endocytosis pathways is the clathrin - mediated endocytosis , which is responsible for the uptake of a wide variety of macromolecules from extracellular environment.(27 ) to quantify the uptake of sirna lipoplexes through this pathway , bsc cells were treated with chlorpromazine or cytochalasin d , which inhibit clathrin - mediated endocytosis,(28 ) followed by sirna lipoplex transfection . both inhibitors reduced the uptake of cy5 labeled sirna lipoplexes by 50% ( figure 2a and supplementary figure s2a in the supporting information ) . however , sirna mediated knockdown of cyb transcript was not diminished in the presence of these inhibitors ( figure 2b ) . inhibition of clathrin - mediated endocytosis significantly blocks uptake of sirna lipoplexes but not functional sirna delivery . bsc cells were treated with chlorpromazine ( chlorp , 10 g / ml ) or cytochalasin d ( cytod , 100 m ) for 1 h followed by sirna lipoplex transfection for 4 h in the presence of the inhibitors . after washing , sirna lipoplex uptake was measured by flow cytometry ( see   for details ) . transfected cells were cultured for another 20 h before cyb transcript level was measured by real - time pcr . ( b ) relative levels of cyb transcript in the presence or absence of inhibitors as normalized to the level of untreated cells . ( c ) relative levels of cyb transcript in bsc cells that expressed the wild - type ( wt ) or k44a dynamin . bsc cells that stably express either the wt or k44a dynamin were transfected with sirna lipoplexes for 4 h , washed , cultured for 20 h , and then assayed for cyb transcript level . dynamin is required for the clathrin - mediated endocytosis . to confirm the results with pharmacological inhibitors , we constructed bsc cells that constitutively expressed the wild - type or a dominant - negative form of dynamin with mutation of lysine at the position 44 to alanine ( termed k44a).(29 ) expression of the mutant but not the wild - type dynamin inhibited the uptake of transferrin ( supplementary figure s2b in the supporting information ) , a marker for the clathrin - mediated endocytosis . however , sirna - mediated knockdown of the target transcript was not affected by expression of either the wild - type or k44a dynamin ( figure 2c ) . similarly , transient expression of k44a dynamin in human embryonic kidney cells 293 ft did not inhibit the knockdown of target transcript following sirna lipoplex transfection ( supplementary figure s2c in the supporting information ) . inducible expression of k44a dynamin in human cervical carcinoma cells hela also did not affect target transcript knockdown ( supplementary figure s2d in the supporting information ) . together , these results show that although the clathrin - mediated endocytosis is a major pathway for cellular uptake of sirna lipoplexes , it does not appear to contribute significantly to the functional sirna delivery . next , we determined if functional sirna delivery is dependent on cholesterol because cholesterol is a critical component of the cytoplasmic membrane and plays a critical role in many membrane - associated events , including membrane fusion , macropinocytosis , and caveolin- and lipid - raft - mediated endocytosis . bsc cells were treated with nystatin or filipin for 1 h to deplete cholesterol from the plasma membrane and then transfected with sirna lipoplexes for 4 h in the presence of the inhibitors . after washing , cells were assayed for sirna lipoplex uptake by flow cytometery or cultured for 20 h and then assayed for cyb transcript level . under these conditions , cytotoxicity was minimal because > 90% of the cells did not take up propidium iodide . as shown in figure 3 , although treating cells with nystatin or filipin had minimal effect on the uptake of sirna lipoplexes , target mrna knockdown was completely abolished in the presence of filipin and significantly reduced in the presence of nystatin . to exclude the possibility that the inhibitors may have interfered with the functional sirna delivery by destabilizing the sirna lipoplexes , we measured the particle size of sirna lipoplexes in the presence of the inhibitors by dynamic light scattering . when the inhibitors were added to sirna lipoplexes , the size of the particles remained the same , with > 95% of the particles were 100200 nm in diameter ( data not shown ) . in addition , after the inhibitors were removed from the sirna lipoplexes suspension by dialysis , the sirna lipoplexes remained active and mediated efficient rnai ( supplementary figure s3 in the supporting information ) . hence , depletion of cholesterol from the plasma membrane abolishes functional delivery of sirna mediated by cationic lipids . ( a ) uptake of sirna lipoplexes in the absence or presence of either nystatin ( 100 g / ml ) or filipin ( 10 g / ml ) as measured by flow cytometry . ( b ) relative cyb transcript level in the presence or absence of the inhibitors . representative data from one of three experiments are shown . among the various endocytic pathways that are sensitive to cholesterol depletion , the caveolin - mediated endocytosis is perhaps the best characterized . to determine whether this pathway contributes to the functional delivery of sirna by cationic lipids , we constructed bsc cells that stably expressed one of the two dominant - negative caveolin constructs : one mutant , termed cav - dn , harbored a deletion of amino acid residues 181 at the n - terminal ; and the other mutant , termed cav - fed , had amino acid residues 6873 replaced with aaaaag.(26 ) expression of the mutant but not the wild - type caveolin in bsc cells resulted in the inhibition of the uptake of cholera toxin ( supplementary figure s4a in the supporting information ) , suggesting that caveolin - mediated endocytosis is inhibited significantly . however , transfection of sirna lipoplexes into bsc cells expressing either the wild - type or the mutant caveolin resulted in cyb knockdown to the same extent ( figure 4a ) . similarly , transient expression of the dominant negative caveolins in 293 ft cells did not diminish cyb transcript knockdown ( supplementary figure s4b in the supporting information ) . functional delivery of sirna lipoplexes is not dependent on caveolin- or lipid - raft - mediated endocytosis or macropinocytosis . ( a ) relative levels of cyb transcript in bsc cells that express the wild - type ( wt ) or the mutant caveolins . bsc cells that stably expressed either the wild - type or one of the mutant caveolins were transfected with sirna lipoplexes for 4 h , washed and cultured for 20 h. the level of cyb transcript was quantified . ( b , c ) effect of pdmp ( b ) or amiloride ( c ) on functional sirna delivery . bsc cells were treated with pdmp ( 10 m ) or amiloride ( 3 mm ) for 1 h , followed by sirna lipoplex transfection for 4 h , washed , and cultured for 20 h. the level of cyb transcript was quantified . ( d ) uptake of cy5 labeled sirna lipoplexes in the absence or presence of inhibitors as quantified by flow cytometry . d , l - threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol ( pdmp ) inhibits synthesis of sphingolipid,(37 ) a component enriched in domains of lipid raft.(38 ) treatment of bsc cells with pdmp had no effect on knockdown of target transcript following transfection with sirna lipoplexes ( figure 4b ) although it inhibited the uptake of sirna lipoplexes by approximately 20% ( figure 4d ) . similarly , amiloride , a strong inhibitor of macropinocytosis,(39 ) showed no significant effect on either the knockdown of cyb transcript or sirna lipoplex uptake ( figure 4c , d ) . together , these results suggest that functional delivery of sirna lipoplexes does not appear to depend on caveolin- or lipid - raft - mediated endocytosis or macropinocytosis . to determine if the functional delivery of sirna lipoplexes is an energy - dependent process , we treated the cells with deoxy - glucose and sodium azide to inhibit atp biosynthesis . bsc cells were treated with both inhibitors for 1 h and then transfected with sirna lipoplexes for 4 h in the presence of the inhibitors . after washing , cells were assayed immediately for sirna uptake or cultured for 20 h in the absence of the inhibitors ( to avoid inhibition of rnai reaction ) and then assayed for cyb transcript level . however , inhibition of atp biosynthesis during transfection did not diminish the target transcript knockdown ( figure 5b ) , suggesting that the functional delivery of sirna lipoplexes does not appear to depend on new atp biosynthesis . ( a ) uptake of sirna lipoplexes in the absence or presence of deoxy - glucose ( 40 mm ) and sodium azide ( 40 mm ) . ( b ) relative level of cyb transcript in the presence or absence of atp biosynthesis inhibitors . ( c ) representative data are shown as mean  sd ( n = 3 or 4 ) . bsc cells were precooled to 4 , 10 , or 20 c for 1 h and then transfected with sirna lipoplexes for 4 h at the same temperatures . after washing , the cells were incubated at 37 c for 20 h and assayed for cyb transcript level . knockdown of the target transcript was significantly reduced at low temperatures ( figure 5c ) . curve fitting suggests that the reduction of target transcript level was exponential ( no sharp transition ) as temperature decreases , suggesting that the effect of temperature on functional delivery of sirna lipoplexes is gradual . in this study , we examined pathways by which sirna lipoplexes are taken up by cells to determine whether specific uptake pathways contribute to functional sirna delivery . our results show that 95% of sirna lipoplexes that enter cells end up in the endolysosomes , most of which are contributed by clathrin - mediated endocytosis ( 50% ) and lipid - raft - mediated endocytosis ( 20% ) . these findings are consistent with observations that endocytosis is the major pathway for the uptake of dna lipoplexes . despite the contribution to the uptake of the majority of sirna lipoplexes , the endocytotic pathways do not appear to contribute significantly to functional sirna delivery as measured by target transcript knockdown . consistent with this observation , while endocytosis is an energy - dependent process , functional sirna delivery mediated by cationic lipids is independent of new atp biosynthesis during the transfection period . furthermore , inhibition of clathrin - mediated endocytosis by either pharmacological inhibitors or expression of a dominant negative dynamin did not inhibit the knockdown of the target transcript ( figure 2 ) . similarly , inhibition of caveolin - mediated endocytosis , lipid - raft - mediated endocytosis or macropinocytosis did not diminish the knockdown of the target transcript ( figure 4 ) . while these results do not exclude the possibility that a small amount of sirna lipoplexes  leaked  out of endolysosomes following endocytosis and contributed to the target gene silencing , taking together , they suggest alternative mechanisms of cationic lipid - mediated functional sirna delivery . the functional delivery of sirna mediated by cationic lipids is shown here to be a rapid process as inferred from the rapid knockdown of target transcript ( figure 1c ) . furthermore , the process is temperature sensitive but does not require new atp biosynthesis during the transfection period ( figure 5 ) . based on these characteristics , the mechanism underlying the cationic lipid - mediated functional sirna delivery is probably through direct fusion between sirna lipoplexes and the plasma membrane . membrane fusion requires cholesterol,(31 ) and it is notable that depletion of cholesterol from the plasma membrane by treating cells with nystain or filipin abolished or significantly inhibited target transcript knockdown ( figure 3 ) . because treating cells with nystain or filipin did not significantly inhibit the bulk uptake of sirna lipoplexes , the functional sirna delivery likely occurs through a minor uptake pathway , consistent with the mechanism of a direct fusion . the mechanisms by which cationic lipids mediate dna delivery have been investigated extensively . during cationic lipid - mediated dna delivery , the lipids function by compacting plasmid dna into dna - containing liposomes or lipiddna complexes ( lipoplexes ) . the lipoplexes facilitate cellular uptake of dna primarily via endocytosis following binding of positively charged lipiddna lipoplexes to the negatively charged cellular surfaces . although fusion of dna lipoplexes with the plasma membrane was initially suggested as a way to deliver dna directly into the cytoplasm,(19 ) lipid mixing assays did not reveal a correlation between fusion of dna lipoplexes with plasma membranes and their transfection efficiency . rather , it seems that when dna lipoplexes fuse with the cellular membrane , dna tends to be excluded at the cell surface and does not enter the cell.(48 ) because the majority of dna lipoplexes end up in the endolysosomes , most of the effort to enhance dna transfection has focused on stimulating the escape of dna lipoplexes from the endolysosomes . what could account for the differences in the mechanisms of cationic lipid mediated sirna and dna delivery ? this similarity could account for the uptake of sirna or dna lipoplexes by the same endocytosis pathways . however , sirna is much smaller than dna and functions in the cytosol whereas dna has to be transported into the nucleus to be transcribed . upon fusion of sirna lipoplexes with the plasma membrane , some sirna is likely delivered directly into the cytosol , where sirna can be readily recruited into rnai mechinary to exert its effect . in contrast , dna delivered into the cytosol via fusion has to be transported into nucleus before it can be transcribed . even if some dna is delivered directly into the cytosol , it may not contribute significantly to the functional dna delivery . this difference could potentially account for the different mechanisms by which cationic lipids mediate functional sirna versus dna delivery . thus , one practical significance of our observations is that optimizing conditions for fusion between sirna lipoplexes and the plasma membrane should improve functional sirna delivery by cationic lipids . in summary , our studies show that although most sirna lipoplexes enter cells via endocytosis , this mode of entry does not appear to contribute significantly to functional sirna delivery . instead , a minor but rapid pathway , probably mediated by fusion of sirna lipoplexes with the plasma membrane , accounts for the finding that only a small fraction of the sirna lipoplexes that enter cells are responsible for most of the observed sirna - mediated target gene knockdown . rnai , rna interference ; sirna , small interfering rna ; cytod , cytochalasin d ; chlorp , chlorpromazine ; cyb , cyclophilin b ; gfp , green fluorescent protein ; pdmp , d , l - threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol .