the specific treatment of cyanide ( cn ) intoxication means the use of scavengers ( e.g. , methemoglobin former sodium nitrite ( sn ) or cobalt compounds or cyanohydrin formers , hydroxocabalamin ( cyanokit has been approved in the us ) , cobinamide , and/or the conversion of cn to the less toxic thiocyanate ( scn ) with exogenously administered sulfane sulfur and sulfurtransferase enzymes [ 24 ] . rhodanese ( rh ) is the best characterized multifunctional , mitochondrial sulfurtransferase [ 58 ] catalyzing the transfer of a sulfane sulfur atom from a donor molecule to cyanide . determining the exact role of nitrite in cyanide antagonism earlier studies were focusing on the methemoglobin - forming effect of nitrite that act as a scavenger by forming a relative stable complex of cyanomethemoglobin [ 3 , 4 ] . very recent studies are focusing on the mitochondria - linked mechanism of nitrite as a nitric oxide donor [ 911 ] . extensive researches are also focusing on developing effective sulfur - containing compounds serving as sulfur donors for reacting with cn with or without rh . thiosulfate ( ts ) is the classical sulfur compound found to participate in the enzyme reaction [ 3 , 4 , 12 ] . however , ts has limited ability to reach the endogenous rh enzyme because of a nearly exclusive extracellular distribution . baskin et al . , reported results on the efficacy of various sulfur donors demonstrating that altering the chemical substituent of the longer chain sulfide modified the ability of the candidate molecule to protect against cn toxicity . earlier investigations were focused on administration of free rh and the sulfur donor ( sd ) directly into the bloodstream [ 1518 ] . unfortunately , the free rh enzyme was rapidly destroyed by the body 's immune system , which makes the efficacy of this approach quite limited . to overcome the limitations for the circulating free rh , micro- or nanosized carrier systems among others sterically stabilized unilamellar liposomes of ~100150  nm diameter are in the focus of recent encapsulation efforts . the encapsulation of rh with a sulfur compound into liposomes  the so - called coencapsulation  can offer further advantages . over stability enhancement for rh the coencapsulation can provide better overall conversion of cn , since the basis component for enzyme reaction , the sulfur donor no longer has to penetrate the liposome membrane . the lipid composition has a significant impact on the encapsulation efficiency of the rh and/or sulfur compound and on the in vivo stability and antidotal effect of the carrier system . thus , optimization of the liposomal composition is an inevitable step in the design of novel antidotal systems . present work deals with a new lipophilic sulfur - containing compound , developed at the us army medical research institute of chemical defense , called dto . in order to achieve the highest cn antidotal protection , the objectives of this study are ( 1 ) optimization of the liposomal encapsulation for the new sulfur donor , dto , with superior sulfur donor reactivity to the present therapy ts ; ( 2 ) in vivo efficacy study of the coencapsulated dto with rh in combination with sodium nitrite on mice . all chemicals employed were of the highest purity commercially available : potassium cyanide , ts , sodium nitrite , phosphate buffer components , ethanol , sodium chloride , concentrated hydrochloric acid , and sodium hydroxide were purchased from j. t. baker , ( phillipsburg , nj ) , formaldehyde and ferric nitrate were purchased from fisher scientific ( pittsburgh , pa ) . the liposome components ( 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine ( popc ) , 1 , 2-dioleoyl - sn - glycero-3-phosphocholine ( dopc ) , n-[1-(2,3-dioleoyloxy)propyl]-n , n , n - trimethylammonium chloride ( dotap ) , soy lecithin ( lec ) , cholesterol ( chol ) ) and rh were purchased from sigma - aldrich ( st . louis , mo ) , and 1,2-dipalmitoyl - sn - glycero-3-phosphoethanolamine - n-[methoxy(polyethylene glycol)-2000 ] ammonium salt ( peg - pe-2000 ) was purchased from avanti polar lipids ( alabaster , al ) . bio - rad protein kit was purchased from bio - rad life sciences laboratories , hercules , ca . male ( cd-1 ) mice ( charles river breeding laboratories , inc . , wilmington , ma ) weighing 1820  g were housed at 21c and in light - controlled rooms ( 12  h light / dark , full - spectrum lighting cycle with no twilight ) , and were furnished with water and 4% rodent chow ( teklad hsd , inc . , city , wi ) ad libitum . all animal procedures were conducted in accordance with the guidelines by the guide for the care and use of laboratory animals ( national academic press , 1996 ) , accredited by aaalac ( american association for the assessment and accreditation of laboratory animal care ) . at the termination of the experiments , surviving animals were euthanized in accordance with the 1986 report of the avma panel of euthanasia . popc , dopc , dotap , lec , peg - pe-2000 , and chol dissolved in ethanol were applied in various molar ratios in order to determine the optimal lipid composition . ph = 7.4 ; osmolarity = 290  mosm ) was added to the dry lipid films . four different rh concentrations ( 0.25  mg / ml , 0.50  mg / ml , 1.00  mg / ml and 1.67  mg / ml ) , and four different dto concentrations ( 2.0 ; 10.0 ; 20.0 and 30.0  mm ) were investigated with various liposomal lipid compositions to evaluate the effects of these parameters on encapsulation efficiency for rh and/or dto . the total lipid concentration ( lipids and chol together ) was 10.0  mg / ml . in order to obtain a homogenous population of small unilamellar vesicles ( suv ) , the multilamellar vesicles ( mlv ) were extruded through polycarbonate filters ( 100 and 400  nm ) with an extruder ( avanti polar lipids inc . the formation of scn from cn was measured spectrophotometrically ( genesys 10uv , thermo electron corporation , waltham , ma ) by the method of westley , with minor modifications of petrikovics et al . . one unit of rh was defined as the amount of enzyme that forms 1  mol of scn in 1  min . formation of scn from cn with the investigated sulfur donors of ts and dto were determined spectrophotometrically by the method of westley    with minor modifications of petrikovics et al . . four different rh concentrations ( 0.25  mg / ml , 0.50  mg / ml , 1.00  mg / ml , 1.67  mg / ml ) were employed with a lipid composition of popc  :  chol  :  peg - pe-2000 with and without dotap . optimal lipid composition for rh encapsulation was determined based on the highest enzyme activity achieved by the same encapsulation process with various lipid compositions . unencapsulated rh was separated from sl - rh by gel filtration on a g-100 sephadex gel column ( 0.7  cm  10  cm ; ge healthcare biosciences ab , sweden ) . ( 1)encapsulation  efficiency  ( % )  =  activity  of  sl - rhtotal  rh  activity    100 . 
							 for the spectrophotometric assays , the encapsulation efficiency for the sulfur donor dto was determined by the rh assay described above with constant rh concentration . when rh concentration was constant , the rate of formation of scn was directly proportional to the sulfur donor concentration . ( 2)encapsulation  efficiency  ( % )  = concentration  of  encapsulated  dtototal  dto  concentration  100 . formation of scn by sl - rh - dto with various lipid compositions was measured spectrophotometrically as described above . ( 3)encapsulation  efficiency  ( % )  =  scn  formation  by  the  given  sl - rh - dtoscn  formation  by  the  original  ( before  encapsulation )  rh  and  dto  concentration  100 . experimental animals received kcn after pretreatment with antagonist(s ) ( sulfur donors and/or rh and/or sn ) . freshly prepared sl - dto , sl - dto - ts , sl - dto - rh , and sl - dto - ts - rh were administered intravenously ( iv ) by tail vein injection to mice 10  min prior to receiving cn ( sc ) . using 10  ml / kg doses of ( sl - dto - rh ; table 4 experiment 5 ) or ( sl - dto - ts - rh ; table 4 experiments 6 and 7 ) the animals received 1520 units of rh . employing 10  ml / kg injections of ( sl - dto ; table 4 experiment 2 ) or ( sl - dto - ts ; table 4 experiments 3 and 4 ) the dose for dto was 11.5  mg / kg and 14.2  mg / kg for the coencapsulated ts . sn ( 100  mg / kg ; sc ) , was injected 45  min prior to cn ( sc ) injection ( table 4 experiments 4 and 7 ) . the animals were evaluated 24 hours after cn exposure for mortality ; surviving animals were observed for an additional week for late - developing toxicity . no toxic effects which could be attributed to sl - dto , sl - dto - ts , sl - dto - rh , sl - dto - ts - rh , ts , or sn ( when administered alone or in various combinations ) were noted in any of the mice at the doses applied . ld50 values were determined by the dixon   up and down method , using 818 mice for each ld50 determination . ( apr ) is expressed as a ratio of ld50 ( mean ) of cn with antagonists and ld50 ( mean )   of cn without antagonists . these studies focused on the encapsulation optimization for new sulfur donor dto when encapsulated with rh and/or ts within sterically stabilized liposomes . the in vitro sulfur donor reactivity comparison shows that dto reacts 15-times faster with cn at constant rh concentration than ts ( table 1 ) . encapsulation efficiencies for both rh and dto were optimized as a function of rh - load , dto - load , and lipid composition . when encapsulating rh alone , small amount of the cationic lipid dotap proved to be beneficial to enhance encapsulation efficiency ( table 2 ) . the optimum rh concentration within the liposomes was 0.25  mg / ml ( table 2 ) . for the encapsulation of dto with a concentration of 2  mm , six different liposomal compositions were examined to rule out the role of lipid composition ( table 3 ) . each contained peg - pe-2000 in 5.1  mol% , lipid - to - chol ratio was 9 to 1 . also , the cationic lipid , dotap in 3.0  mol% , was utilized to influence the charge of the liposomal surface in hopes that a positively charged surface would provide increased affinity for dto . on the basis of the results it can be concluded that the presence of dotap leads to significant ( p < 0.05 ) higher encapsulation efficiency with dopc and popc as main lipid component . lec liposomes demonstrated the least encapsulation efficiency for dto , and dotap appeared to provide little to no enhancement ( table 3 ) . the role of dotap in enhancing the encapsulation efficiency can be explained with electrostatic interactions between dotap and dto at the ph value examined ( ph = 7.4 ) . early experiments with the 60  :  40 ratio of dipalmitoyl - phosphatidylcholine ( dppc ) to chol composition provided very low encapsulation efficiency for encapsulation of dto ( data not shown here ) . as dto is a lipophilic sulfur donor , it can be expected that it is localized in the liposomal bilayer , more or less immersed in it . the saturated bonds of dppc providing an ordered , relative rigid structure may inhibit the immersion of dto in the liposomal membrane . contrarily , popc and dopc possessing double bonds and as a consequent of it a less ordered and more fluid membrane structure can promote the encapsulation of dto . thus , instead of dppc , dopc , or popc were used for liposome preparation and ratios were adjusted to 90  :  10 lipid to chol . the results shown in table 3 indicate that dopc liposomes containing 3  mol% dotap provided the highest encapsulation efficiency at 81.7  3.1% . popc liposomes with 3% dotap were close behind with an encapsulation efficiency of 78.4  2.3% . however , there was no significant difference between encapsulation efficiencies with dopc and popc . the liposome compositions including dotap were used in further experiments due to the increase in encapsulation efficiency achieved by these films . the effect on encapsulation efficiency by the increase in dto concentration was evaluated for dopc and popc containing liposome compositions with both sets of liposomes containing 3% dotap . in order to evaluate the role of dto concentration on the encapsulation efficiency each set 's films were prepared with dto concentrations of 10  mm , 20  mm , and 30  mm . the encapsulation efficiency remained high for each liposome formulation containing 3% dotap for each applied dto concentrations of 10  mm , 20  mm , and 30  mm . the encapsulation efficiencies of dto for popc samples were 69.7  2.3% , 82.8  7.1% , 79.2  8.1% , while for the dopc samples dto encapsulation efficiencies of 74.2  2.0% , 86.2  3.9% , and 89.9  4.2% were determined , for 10  mm , 20  mm , and 30  mm dto concentrations , respectively . for a given dto concentration there was no significant difference between the encapsulation efficiency values for dopc or popc liposomes ( p > 0.05 ) . the optimal liposome composition for the encapsulation of rh was determined in earlier experiments to be the 60  :  40 ratio of popc to chol . however , the prior experiments did not evaluate the dopc or the cationic lipid dotap . furthermore , rh was added either in isotonic hepes buffer ( ph = 7.47.7 ) or in 5% ( w / w ) aqueous solution of glucose ( glu ; ph = 4.27.8 ) to the dry lipid films . for the purpose of coencapsulating dto with rh , the rh encapsulation efficiency must be determined for the same lipid compositions and in the same hydrating systems as in the case of dto . the optimal liposome composition for rh encapsulation was the 90  :  10 ratio of popc to chol with the use of dotap . also , the 3.0  mol% dotap again increased the encapsulation efficiency for most of the different liposomal compositions ( table 2 ) .   for the coencapsulation of dto and rh , the combination of popc , chol , peg - pe-2000 , and dotap ( with molar ratios of 82.7  :  9.2  :  5.1  :  3.0 ) was chosen as the most adequate liposome composition . the mentioned composition of sterically stabilized , positively charged liposomes performed the best in the coencapsulation , with a coencapsulation efficiency for rh and dto of 88.6  17.1% ( with a rh load of 0.25  mg / ml and a dto concentration of 30  mm ) . as the coencapsulation efficiency was determined on the basis of scn formation by sl - rh - dto ; the given value represents the combined effect of rh and dto in cn conversion . for the sake of comparison , encapsulation efficiency for the coencapsulated rh alone  with 0.25  mg / ml concentration  was 74% , while for dto alone  with 10  mm dto load  was 57.7  8.1% . increasing the concentration of dto produced similar encapsulation efficiencies , than in case of 10  mm . with dto loads of 20  mm and 30  mm for the coencapsulated dto encapsulation efficiencies of 55.6  4.0% and 61.6  17.6% were measured , respectively . the conversion of cn to scn by the coencapsulation of 10  mm , 20  mm , and 30  mm dto with rh also proved to remain linear with an r value of 0.9930 . the ability to co - encapsulate dto , or any other sulfur donor molecule with rh should provide better overall conversion of cn , since the sulfur donor no longer has to penetrate the liposome membrane . 
 in vivo evaluation of the optimized liposomal preparations made from dto/(dto + ts ) and/or rh were tested as cyanide antidotes on a mice model . based on the above optimization efforts , for further in vivo evaluations we employed 3% dotap ; 0.25  mg / ml rh load , 30  mm dto load with the lipid composition of popc  :  chol  :  peg - pe-2000  :  dotap = 82.7 : 9.2 : 5.1 : 3.0 . the in vivo prophylactic treatment results with rh and dto / ts encapsulated within the optimized liposomal formulations are shown in table 4 . this protection was enhanced ( apr = 4.8 ) when ts was coencapsulated with dto in a molar ratio of 1  :  1 ( table 4 experiment 3 ) . coencapsulation of ts with dto is also believed to provide protection against product inhibition by sulphite ( zottolla , personal communication ) . as it was expected , sn further enhanced the protection with the apr of 6.7 . when dto was coencapsulated with rh ( table 4 experiment 5 ) , the apr was 3.9 . when dto was coencapsulated with ts and rh , ( sl - dto - rh ) the apr was enhanced to 4.9 ( table 4 experiment 6 ) . the highest protection ( apr = 15.3 ) was achieved with the combination of ( sl - dto - ts - rh ) and sn ( table 4 experiment 7 ) . expressing the relative antidotal potency ratios ( rapr ) better indicates the differences in protection with two antidotal systems ( table 5 ) . comparing experiment 2 and 3 ( rapr = 2.2 ) represents the effects of ts coencapsulation with dto . when comparing experiments 3 and 4 ( rapr = 1.4 ) or experiments 6 and 7 ( rapr = 3.1 ) the enhancement effects reached by sn are represented . the significant enhancement by rh is expressed when comparing experiments 2 and 5 ( rapr = 1.8 ) and experiments 4 and 7 ( rapr = 2.3 ) confirming earlier studies with other types of encapsulation formulations for rh [ 23 , 26 , 27 ] . table 6 shows the therapeutic antidotal protection with the ( sl - dto - ts - rh ) combinations with and without sn . at approximately 2 ld50 dose of kcn ( 15  mg / kg ) , all the 6 animals survived in each experiment ( table 6 experiments 1 , 2 , and 3 ) . however , when the kcn dose was enhanced ( 20  mg / kg , approximately 3 ld50 ) the survival rate with ( sl - dto - ts ) + sn was 67% ( table 6 experiment 4 ) , while with ( sl - dto - ts ) without sn provided a 50% survival rate ( table 6 experiment 6 ) . however the ( sl - dto - ts - rh ) antidotal system with and without sn also provided a 100% therapeutic protection ( table 6 experiments 5 and 7 ) . the present experiments and results are confirming that the approach of utilizing externally administered , encapsulated , metabolizing rhodanese may have broad implications in cyanide antidotal therapy . the application of this approach has been successfully tested in animal models . in summary , these studies are describing the prophylactic and therapeutic in vivo efficacy of the encapsulated rh and the new , reactive sulfur donor dto . optimization efforts were attempted for the liposomal lipid compositions , rh - load , and coencapsulation of two sulfur donors ( ts and dto ) and rh to enhance the encapsulation efficiency for the given components . optimization of the carrier systems is always a major part of these types of research efforts . considering the high lipophilicity of dto , for further in vivo applications other introduction routes ( e.g. , intramuscular ) with further formulation optimizations are recommended .