several emerging rna viruses have been the cause of international epidemics within the past few years . the four dengue virus ( denv ) serotypes have considerably expanded their geographic distribution in recent years . with billions of people at risk , more than 50 million cases , and 1250025000 deaths annually , , the presence of four denv ( denv 1 , 2 , 3 , and 4 ) serotypes has complicated the design of vaccines because incomplete protection against one serotype may influence the disease outcome once infection is established by a different serotype , through a process referred to as antibody - mediated disease enhancement . since no specific antiviral treatment is available for flaviviruses , there is an urgent need for antiviral drugs . flaviviruses are enveloped viruses   with single - strand positive - sense rna genome of about 11  kbs . there are more than 80 different flaviviruses   and most of them are important human pathogens , such as yellow fever virus ( yfv ) , west nile virus ( wnv ) and its australian variant kunjin virus ( kunv ) , japanese encephalitis virus ( jev ) , tick - borne encephalitis virus ( tbev ) , saint louis encephalitis virus ( slev ) , and denv . the c - terminal domain of nonstructural protein 3 ( ns3 ) has an atp - dependent helicase activity that maintains viral replication and for that reason is a good target for designing selective inhibitors of viral replication as therapeutic intervention . identified the widely used antihelminthic drug ivermectin as a molecule able to inhibit the ns3 helicase activity of several flaviviruses in vitro at submicromolar concentrations . most importantly , ivermectin proved to be a selective inhibitor of the replication of yfv   and , although less efficiently , of other flaviviruses such as denv , jev , and tbev . ivermectin is now under clinical trials on humans , as a denv therapeutic , at mahidol university in thailand . ivermectin is the common name of 22,23-dihydroavermectin b1 , a semisynthetic derivative of avermectin family . it is a potent endo- and ectoparasitic agent with a broad spectrum of activity ; its clinical use has been greatly recognized up the top award in science , being the nobel prize in medicine and physiology 2015 awarded to william c. campbell and satoshi mura for their discovery of avermectin . the pharmacokinetic behavior of avermectin depends upon the route of administration , the formulation used , the animal species , and pathophysiological status of the host . it is established that subcutaneous injection is the most efficient route for ivermectin administration in terms of drug bioavailability in sheep , cattle , and goats when compared with oral and topical administration . pharmaceutical technology has been applied to develop different drug formulations and delivery systems to optimize the pharmacological availability of ivermectin . the most promising approach for improving formulation lies in innovative delivery systems using carriers with defined physicochemical properties , such as liposomes , microemulsion , and polymeric micelles . although the efficacy of ivermectin has been established in humans against several parasite diseases , the antiviral properties of this compound are not yet exploited , mainly due to its complex chemical structure which can not be easily chemically modified . the lack of appropriate formulations which could improve cellular internalization of ivermectin reduces the unfavorable effect of the drug . according to these considerations , the current paper reports the development of engineered liposome formulations for possible clinical use of ivermectin . in particular , this study describes the production and the characterization of liposomes with ivermectin followed by subsequent tests of their antiviral activity and cytotoxicity in different cell lines . our results show that ivermectin , when delivered through liposomes , reduces the cytotoxicity and , at the same time , inhibits denv replication with ec50 values in the micromolar range improving the performance of ivermectin alone . highly pure phosphatidylcholine ( pc ) 90% from soybean ( phospholipon 90 g lipoid , germany ) ; cholesterol 97% ( fluka , germany ) ; dimethyldioactdecylammonium bromide ( ddab ) ( sigma - aldrich , uk ) ; dimethyldioctadecylammonium chloride ( ddac ) ( sigma - aldrich , uk ) ; and ivermectin ( sigma - aldrich , uk ) are used . the determination of entrapment efficiency was conducted with sepharose 4b ( pharmacia , uppsala , sweden ) and isotonic palitzsch buffer ( ph 7.44 ) as previously described . for tlc analysis , iodine was used ( fluka , germany ) . all the other reagents and solvents were from sigma - aldrich , uk , having analytical grade . typically , ivermectin was solubilized in ethanol ( final concentration 50  mm ) and different volumes were mixed with an ethanolic solution of pc 30180  mm , plus cholesterol ( 10  mm ) or ddab ( 510  mm ) , in order to obtain the final required ivermectin concentrations . 500  l of the resulting ethanolic solution was injected by a syringe pump ( 500  l / min ) into 4.5  ml of double - distilled water under magnetic stirring ( 300  rpm ) . liposome size analysis with photon correlation spectroscopy ( pcs ) , the cryogenic transmission electron microscopy ( cryo - tem ) analysis , and the determination of encapsulation efficiency were performed as previously described . all the cell lines were grown at 37c in a humidified atmosphere of 5% co2 with the addition in the medium of 100  u penicillin / ml and 100  g / ml streptomycin . african green monkey kidney epithelial cells 118 ( vero-118 ) were grown in imdm ( life technologies ) supplemented with 10% or 1% fetal bovine serum ( fbs ) . mouse leukaemic monocyte macrophage cells ( raw 264.7 ) were grown in dmem ( life technologies ) supplemented with 10% or 2% fbs , 2  mm l - glutamine , 20  mm hepes , 0.075 african green monkey kidney epithelial cells f ( vero - f ) and baby hamster kidney ( bhk ) cells were cultured in dmem ( life technologies ) containing 10% or 2% fbs . hepatocellular carcinoma cells ( huh-7 ) were grown in dmem ( life technologies ) containing 10% fetal calf serum ( fcs ) . dengue virus strains were obtained from the early dengue infection and outcome ( eden ) study in singapore   with the following genbank accession numbers : denv 1 ( eu081230 ) and denv 2 ( eu081177 ) [ 13 , 14 ] . cells were seeded ( 2  10  cells / well ) in 96-well plates either in the absence ( controls ) or in the presence of ivermectin ( or 2-cmc taken as reference antiviral compound ) at different concentration ( 0.225.0  m ) . after 4 days of cell culture at 37c ( 35c for vero-118 ) , the medium of each well was removed . 200  l of ethanol 70% was added to all wells and after overnight incubation it was removed . 50  l of filtered 1%  ( w / v ) methylene blue in pbs was added to each well . the wells were analysed giving a score to each well ( high value : white ; low value : blue ) . cytotoxicity was determined using the mts [ 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h - tetrazolium ] assay . to this aim , the cells were seeded ( 5  10  cells / well ) in 96-well plates either in the absence ( controls ) or in the presence of ivermectin at different concentration ( 0.225.0  m ) . after 4 days of cell culture at 37c a mts / phenazine methosulphate ( pms ) stock solution ( 2  mg / ml mts ( promega ) and 46  g / ml pms ( sigma - aldrich ) in pbs at ph 6.06.5 ) was diluted 1/20 in mem ( life technologies ) . to each well , 75  l of mts / pms solution was added and the optical density ( od ) was read at 498  nm 2  h later . the percentage of cell viability was calculated as ( odtreated / odcc )  100 , where odcc is the od of uninfected untreated cells and odtreated are uninfected cells treated with compound . the half maximal cytotoxicity concentration ( cc50 ) was defined as the compound concentration that reduces the number of viable cells by 50% and it was calculated based on the nonlinear regression dose response sigmoidal curve drawn in graphpad prism . 10 cells were infected with denv 1 , denv 2 , or denv 2 mouse adapted s221 strain at moi 0.3 , in the presence of either ivermectin alone or ivermectin contained in liposomal formulations for 1  h. inocula were discarded and replaced with maintenance media containing the same compounds and kept for 48  h. after 2 days , cell morphology was observed under microscope before collecting supernatant that was used for plaque quantitation assay . the virus titer without drug treatment routinely reached more than 10  pfu / ml , representing the standard infection level in huh-7 cells . half maximal effective concentration ( ec50 ) was defined as the compound concentration that reduces the virus effect by 50% and it was calculated based on the nonlinear regression dose response sigmoidal curve drawn in graphpad prism . the preparation of liposomes was accomplished by ethanol injection since this method allows single - step formation of liposomes with a narrow size distribution , without degradation or oxidation of lipids or drugs . we used the  controlled  injection method in which the ethanolic solution of lipids is injected into the water phase via a syringe pump at a controlled rate , allowing precise ethanol diffusion in water . the mixing of ethanol with water is indeed the crucial step leading to lipid molecule self - assembly , driving liposome formation . in order to optimize the formulation of liposomes for ivermectin ( figure 1(a ) ) , we explored the roles of different experimental parameters on liposome characteristics , such as ( a ) lipid composition , ( b ) type of cationic surfactant , ( c ) pc content , and ( d ) ivermectin content . table 1 reports the complete composition of produced liposomes together with a unique identification code for each formulation . figure 2 summarizes the results of photon correlation spectroscopy ( pcs ) measurements of liposomes composed of neutral lipids ( pc / cholesterol ( a ) ) and cationic lipids ( pc / ddab ( b ) ) . the effect of different amount of ivermectin ( from 0.1 to 1.0  mm ) on the dimension of liposomes is also shown in figure 2 . the obtained results clearly indicate that cationic liposomes are smaller than respective neutral formulations ; for instance , cationic liposomes containing 1.0  mm ivermectin have a z - average of 72  nm , while for the neutral ones z - average is 193  nm . this effect is due to molecular or physical interactions in conjunction with an increase in membrane fluidity induced by ddab . these interactions could promote size reduction by decreasing the radius of curvature of lipid bilayer as a result of phospholipid - cationic head group contact . since the performance of liposomes , such as drug release or cell uptake , is influenced by morphological characteristics , an accurate microscopic characterization is mandatory before any biological evaluation . this technique enables the imaging of liposome architecture to evaluate its morphology together with an estimate of liposome size . the cryo - tem images reported in figure 3 demonstrate that both empty ( ( a ) , ( c ) ) and ivermectin - loaded liposomes ( ( b ) , ( d ) ) , obtained by the  controlled  ethanol injection , had a spherical shape . the presence of a limited number of oligolamellar vesicles ( typically 24 lamellae ) in neutral liposome samples is evident ( ( a ) and ( b ) ) . microscopic images confirm that cationic liposomes are generally smaller than neutral ones and almost entirely composed of unilamellar vesicles with a size smaller than 100  nm in diameter ( in agreement with pcs analysis ) . as further characterization , the effect of pc content on liposome dimension in the produced ivermectin - loaded liposomes was considered . figure 4 shows that an increase of pc from 3  mm to 9  mm causes the growth of the z - average of neutral liposomes from 65  nm to 193  nm and , in case of cationic liposomes , from 30  nm to 72  nm . the loading efficiency of liposomes for ivermectin was determined by size exclusion chromatography . as an example , the elution profile of ivermectin - loaded liposomes ( # pc9-ch1-ive1.0 ) , obtained after sepharose 4b chromatography , is shown in figure 5 . the peak ( indicated by the solid arrow ) contains ivermectin - loaded liposomes ; no peak was detected for free ivermectin , indicating that almost all ivermectin was associated with the liposomes . the presence of ivermectin in liposome peak was demonstrated by recording the uv spectrum of the corresponding fractions at 253  nm . therefore , the ivermectin loading yield was estimated by comparing the absorption at 253  nm of isolated liposomes with ivermectin standard solutions . the yield of ivermectin loading for liposomes prepared with both 3  mm and 9  mm pc was almost higher than 98% ( data not shown ) . such result indicates that the high liposome loading efficacy is achieved when ivermectin liposomes were produced by  controlled different cell lines were treated with several concentrations of the drug ( 0.225  m ) . ivermectin on bhk and raw cells shows cc50 < 1.5  m ( bhk cc50 = 1.5  m , raw cc50 = 0.8  m ; table 2 ) and on vero-118 , vero - f , and huh-7 has higher cc50s ( vero-118 cc50 = 5.7  m , vero - f cc50 = 23.6  m , and huh-7 cc50 = 7.3  m , table 2 ) ( figure 6(a ) ) . in comparison to 2-cmc , already known viral polymerase inhibitor   ( figure 1(b ) ) ivermectin is 10 times more cytotoxic ( figure 6(b ) ; table 2 ) . in order to verify the cytotoxicity of ivermectin carried by liposomes , we tested # pc9-ch1-ive1.0 , # pc9-br1-ive1.0 , # pc9-br0.5-ive1.0 , # pc18-br1-ive1.0 , and the corresponding empty liposomes ( # pc9-ch1 , # pc9-br1 , # pc9-br0.5 , and # pc18-br1 ; table 1 ) on vero - f ( figure 7 ) , vero-118 ( figure 8) , bhk ( figure 9 ) , and raw ( figure 10 ) cell lines . ivermectin in liposomes shows reduced cytotoxicity in comparison to free ivermectin in all the cell lines . the highest effect is shown in vero-118 with cc50 reduction of more than 5 times ( from 5.7  m to more than 25.0  m ) . in bhk and raw cells # pc9-ch1 , # pc9-br1 , and # pc9-br0.5 reduced the ivermectin cytotoxicity 3 times ( from 1.5  m to 4.3  m and from 0.8  m to 2.2  m , resp . ) while # pc18-br1-ive1.0 is completely noncytotoxic within the amount tested ( table 2 ) . # pc3-br1-ive0.1 , # pc3-ch1-ive0.1 , # pc3-cl1-ive0.1 , # pc9-ch1-ive1.0 , # pc9-br1-ive1.0 , and the corresponding empty liposomes were tested on huh-7 . # pc3-br1-ive0.1 is not cytotoxic in the concentration range tested ( from 0.12  m to 10.0  m ) whereas cytotoxicity around 10.0  m was observed for the others after 48  h of incubation . in conclusion all the engineered liposomes are less toxic than ivermectin alone while the empty liposomes have no effect on cells cytotoxicity . the most important is the use of aqueous solvent to solubilise the liposomes instead of the organic solvent that is necessary for ivermectin solubilisation . the antiviral activity of ivermectin , empty liposomes , and ivermectin liposomes was assayed in vitro on different virus strains , namely , denv 1 ( eu081230 ) , denv 2 ( eu081177 ) , and the mouse - adapted strain denv 2 s221 [ 13 , 14 ] . since dengue virus causes systemic infection and in particular the liver is implicated in humans , for the in vitro test , the huh-7 cells were employed , representing one of the standard cell lines that are routinely used for the cell - based assay for dengue infection . .   demonstrated that slight differences in formulation could change the plasma kinetics and efficacy of ivermectin in rabbits , subcutaneously given 0.3  mg / kg of ivomec ( a commercial formulation of ivermectin ) or ivermectin in liposomes . in order to test this finding , the in vitro antiviral activity of free ivermectin was compared to that of different liposomal formulations containing the drug . the antiviral assay was determined by adding the virus and ivermectin or ivermectin in liposomes at the same time . particularly , it is important to underline that the routine setup commonly used for antiviral assays is indeed cotreatment ( i.e. , adding the compound to be tested concomitantly with the virus ) ; thereafter drug / virus inoculum was removed and a further addition of the compound was made for 48  h incubation . by this experimental approach , information about the antiviral activity of the compound ( both direct and indirect acting ) would be obtained . a different order of addition would provide preliminary insights into whether the compound is acting as a viral entry inhibitor ( pretreatment ) or virus replication inhibitor ( posttreatment ) . since ivermectin has been previously shown to act on viral helicase ns3   and/or ns5-importin / , it would be ideal to test the different formulations of ivermectin by cotreatment assay as described in this paper . notably the administration of ivermectin by a liposomal formulation yielded higher cmax and significantly faster absorption . this finding suggested that the use of liposomes could improve the in vivo efficacy of ivermectin . figures 11 and 12 report the activity of liposome preparations against denv 1 , 2 , and s221 on huh-7 cell line . all the three liposome preparations tested against denv 1 ( # pc3-ch1-ive0.1 , # pc3-cl1-ive0.1 , and # pc3-br1-ive0.1 ( figures 11(a ) , 11(b ) , and 11(c ) , resp . ) ) reduced ec50 in comparison to ivermectin alone ( figure 11(d ) ) , with the best results observed for # pc3-br1-ive0.1 , leading to a considerable increase of the antiviral effect ( from 3.7  m to 1.3  m ; figure 11(c ) ) . results ( plain line , figures 11(e ) , 11(f ) , and 11(g ) ) suggest increasing efficacy of liposome packaged ivermectin . # pc3-ch1-ive0.1 ( figure 11(g ) ) resulted in ~2 times reduction in ec50 ( from 2.6  m to 1.3  m , table 3 ) as compared to ivermectin alone ( figure 11(h ) ) , while # pc3-br1-ive0.1 and # pc3-cl1-ive0.1 ( figures 11(e ) and 11(f ) ) have shown great improvement in efficacy from 2.6  m to 0.3  m ( 5 times reduction in ec50 ) . with low cytotoxicity ( > 10.0  m ) and significant antiviral activity ( 0.3  m ) , # pc3-br1-ive0.1 demonstrates being a good candidate for future in vivo experiments . figure 11 also shows the results for liposomes preparation against s221 mouse adapted strain ( dashed lines ) . the respective empty liposomes have no effects on cell lines . in order to analyse a higher concentration of ivermectin ( 1.0  mm ) , we tested liposomes # pc9-ch1-ive1.0 and # pc9-br1-ive1.0 and their respective empty liposomes ( without ivermectin inside ) ( figure 12 ) on huh-7 infected with denv 2 . empty liposomes do not have any effect on viral replication except at phosphatidylcholine concentrations > 300  m . the dose response curve of infected cells treated with ivermectin liposomes is shifted to the left with respect to that of empty liposomes , confirming that ivermectin liposomes are able to reduce the plaque number , and so the active viruses . the liposomes containing 1  mm ivermectin slightly reduce ec50 when compared to free ivermectin ( table 3 ) but there is no significant difference in ec50 with respect to the previous tested liposomes ( # pc3-br1-ive0.1 , # pc3-cl1-ive0.1 , and # pc3-ch1-ive0.1 ) containing 1.0  mm ivermectin . in conclusion we tentatively attribute the increase of antiviral activity ( measured in terms of ec50 on denv 2 ) from 2.6  m to 0.3  m to the fact that liposomes containing ivermectin could merge with cell membranes , facilitating the intracellular uptake of the drug . this hypothesis is supported by the fact that positively charged liposomes ( i.e. , those containing the cationic surfactants ddac and ddab ) such as # pc9-cl1-ive0.1 and # pc9-br1-ive0.1 are the formulations displaying the highest antiviral activity ( ec50 0.3 and 0.6  m , resp . ) . we demonstrate that ivermectin , when delivered through liposomes , reduced cytotoxicity up to 5 times . they can effectively inhibit denv replication with ec50 values in the same range of ivermectin alone and even improve its activity in several formulations . the possibility of dissolving ivermectin into an aqueous solution thanks to the use of liposome as drug carriers is a new step towards the solution of its pharmacokinetics problems , in particular its high cytotoxicity . the important improvement of ivermectin activity loading in liposomes with ions compositions creates a promising starting point for a future development of these nanocarriers which one day could be the possible solution to the lack of drugs against flaviviruses .
rna virus infections can lead to the onset of severe diseases such as fever with haemorrhage , multiorgan failure , and mortality . 
 the emergence and reemergence of rna viruses continue to pose a significant public health threat worldwide with particular attention to the increasing incidence of flaviviruses , among others dengue , west nile virus , and yellow fever viruses . 
 development of new and potent antivirals is thus urgently needed . 
 ivermectin , an already known antihelminthic drug , has shown potent effects in vitro on flavivirus helicase , with ec50 values in the subnanomolar range for yellow fever and submicromolar ec50 for dengue fever , japanese encephalitis , and tick - borne encephalitis viruses . 
 however ivermectin is hampered in its application by pharmacokinetic problems ( little solubility and high cytotoxicity ) . 
 to overcome such problems we engineered different compositions of liposomes as ivermectin carriers characterizing and testing them on several cell lines for cytotoxicity . 
 the engineered liposomes were less cytotoxic than ivermectin alone and they showed a significant increase of the antiviral activity in all the dengue stains tested ( 1 , 2 , and s221 ) . in the current study ivermectin is confirmed to be an effective potential antiviral and liposomes , as drug carriers , are shown to modulate the drug activity . all together 
 the results represent a promising starting point for future improvement of ivermectin as antiviral and its delivery .