oral chemotherapy of leishmaniasis consists of administration of antimonials , miltefosine and paromomycin for about three weeks ( sundar and chakravarty , 2013 ) . as a standard anti - leishmanial chemotherapy , amb is currently administered in an inpatient setting , requiring infusion over several hours because of its potential for generating acute nephrotoxicity and haemolysis ( brajtburg et  al . , 1985 , deray , 2002 ) .
amb , an antifungal drug , primarily acts on membrane ergosterol and kills the parasite by disrupting the surface membrane ( matlashewski et  al . , 2011 ) .
drug resistance to antimonials is spreading , and inclusion of liposomal amphotericin b ( amb ) in the treatment is increasingly recommended ( balasegaram et  al . , 2012 ) .
even in liposomal form , amb is administered by slow intravenous infusion , because of its toxicity ( gahart et  al . , 2016 ) .
this is an inpatient procedure , and represents additional burden in the resource - poor settings where visceral leishmaniasis is prevalent . to our knowledge , there are no new drugs for visceral leishmaniasis in the drug discovery and development pipeline , but several reports are available on anti - leishmanial agents incorporated in particulate delivery systems for use against experimental visceral leishmaniasis ( costa lima et  al . , 2012 ,
kansal et  al . , 2012 , lopes et  al . , 2012 ,
there is scope to improve upon such delivery systems to further ameliorate adverse drug effects , prevent emergence of drug resistance , adapt systems to outpatient use , and reduce cost to patients ( jha , 2006 ) .
we therefore asked whether anti - leishmanial therapy may be augmented with a novel pharmacophore , employed in unrelated diseases , but relevant to the objective of killing the parasite within the macrophage .
no is one such molecule and its role in innate immune responses of mammalian macrophages is well known ( ascenzi et  al . , 2003 , klink and sulowska , 2007 , brune , 2010 , mills , 2012 , bogdan , 2015 ) .
the differences in no cytotoxicity across evolutionarily distinct genera suggest that it can effectively kill bacteria , fungi and protozoa at concentrations that can be tolerated by mammalian cells ( dzik , 2014 ) .
bactericidal activity of no against a number of human pathogens has been extensively reported , and our group has demonstrated the activity of no prodrugs against the macrophage - borne pathogen mycobacterium tuberculosis ( verma et  al . , 2012 , verma et  al . , 2013 ) .
no donors , also termed diazeniumdiolates or nonoates , are formed by the chemical reaction of no with nucleophilic amines ( keefer and saavedra , 2002 ) and can release no in a sustained manner .
no donors are essentially prodrugs , since the active moiety , no , is a gaseous molecule with a biological half - life ( t1/2 ) of not more than 2  min ( liu et  al . , 1998 ) . no donors like deta / no , isosorbide mononitrate , sodium nitroprusside ( snp ) , 3-morpholinosydnonimine ( sin-1 ) , s - nitrosoglutathione ( gsno ) and s - nitroso- n - acetyl penicillamine ( snap ) are familiar pharmacological agents .
deta / no has a significantly longer half - life ( t1/2 ) of 20  h  at ph 7.4 and 37  c ; and releases two moles of no per mole of parent compound without prior biotransformation ( yamamoto and bing , 2000 , keefer and saavedra , 2002 , xu et  al . , 2011 ) .
no has efficacy against leishmania and the effective use of no donors in patients of cutaneous leishmaniasis through topical application of creams containing snap has been demonstrated ( lopez - jaramillo et  al . , 1998 ) .
no - releasing diazeniumdiolates have also been formulated as particles for topical treatment of cutaneous leishmaniasis ( moreno et  al . , 2014 ) .
particulate drug delivery systems are taken up by macrophages , which also represent the ecological niche for amastigotes of lesishmania species that cause visceral leishmaniasis . while the promastigote is killed relatively easily by drugs in blood circulation , the intracellular amastigote survives in phagosomes of tissue - resident macrophages .
the phagosome membrane acts as an additional barrier to the entry of parasiticidal drugs circulating in the bloodstream .
phagosome - resident amastigotes also evade innate host defense mechanisms ( sacks and sher , 2002 ) and interfere with development of defensive immunity .
cytokines such as gamma - interferon activate macrophages to produce no which kills intracellular leishmania ( gatto et  al . , 2015 ) .
mice deficient in inducible no synthase ( inos ) are more susceptible to leishmania infection ( green et  al . ,
several membrane molecules of leishmania inhibit macrophage inos as part of the parasite 's survival strategy inside macrophages , and cells expressing high levels of inos are resistant to leishmania infection ( proudfoot et  al .
we employed a prodrug - in - particle approach to target no to the macrophage cytosol through passive internalization .
the objective of the present work was to establish  proof of principle'in respect of the efficacy of no against visceral leishmaniasis .
our approach also involves incorporating multiple drugs , amb and deta / no in the same particle ( mi et  al . , 2013 ) .
we expected that no delivered to the macrophage cytosol will have important outcomes , both in terms of killing the parasite as well as stimulating the host to mount defense responses .
our observations suggest that the prodrug - in - particle approach may be of use in targeting no to infected macrophages , where the molecule exhibits parasite killing in  vitro and in  vivo , without undue toxicity to the host cell .
biodegradable poly(lactic - co - glycolic acid ) ( plga ) of monomer ratio 65:35 was purchased from birmingham polymers inc . , ( birmingham , al , usa ) .
deta / no , cell culture medium ( rpmi-1640 ) , fetal bovine serum ( fbs ) , supplements and antibiotics ; pluronic f-68 ( poloxamer 188 ) ; methylthiazoletetrazolium ( mtt ) and dialysis tubing ( 70  kda ) were purchased from sigma 
acetone and methanol were of spectroscopic grade and all other reagents and chemicals were of analytical grade .
the water used in all experiments was prepared in a three - stage millipore milli - q plus 185 purification system ( bedford , ma , usa ) .
particles were prepared by a solvent displacement method ( pandya et  al . , 2011 , verma et  al . , 2011 ) .
plga was dissolved in acetone by stirring at 37  c for 10  min to obtain a 2.5% ( w / v ) solution .
methanol containing 4  m sodium iodide was used to solubilize amb to obtain a 0.1% solution ( lopes et  al . , 2014 ) .
deta / no was added to a final concentration of 0.1% and the ph was adjusted to 8.5 .
then , 25  ml of the organic solution were added dropwize into 40  ml of distilled water containing 40  mg poloxamer 188 under homogenization ( ika ultra turrax model t-25 ) at room temperature . the preparation
was then placed in a rotary evaporator at 5558  c under vacuum to evaporate the organic phase and concentrate the aqueous phase to 10  ml .
the aqueous preparation was centrifuged at 70,000  rpm for 30  min and the pellet lyophilized to obtain particles in the dry state .
particle morphology was studied using a scanning electron microscope ( quanta 200 , fei , oregon , usa ) .
three batches were analyzed for size distribution ( mean diameter and polydispersity index ) and zeta potential using a zetasizer nanozs ( malvern instruments , uk ) .
amb content and encapsulation efficiency were determined by a validated hplc method , while the estimation of deta / no was done by uv photometry of the ultracentrifugation supernatant .
each sample of drug loaded particles ( 2  ml ) was filled in dialysis bags with a molecular mass cut - off of 70  kda .
the bags were suspended in 200  ml of 0.1  m phosphate buffer ( ph 7.4 ) containing 1% v / v tween 80 , at 37  c in a usp dissolution apparatus type ii ( disso-2000 , labindia , mumbai , india ) running at 50  rpm . at predetermined intervals , aliquots of 0.5  ml were withdrawn and the amount of drug released estimated by hplc ( verma et  al . , 2011 ) .
uptake of particles was assessed in j774 a.1 mouse macrophages using flow cytometry ( facs aria , bd biosciences , heidelberg , germany ) .
cells were maintained in dmem supplemented with 10% fbs and 1% antimycotic - antibiotic mixture at 37  c , 5% co2 , and 95% relative humidity .
one million cells were plated per well in six - well plates , incubated overnight and washed with incomplete medium to remove non - adherent and dead cells .
particles containing fitc alone ( blank particles ) , or fitc in addition to amb , deta / no alone , or both drugs in the same particle were added to the designated wells at drug concentrations of 50  g / ml or fitc concentration 0.1  g / ml .
after 3  h of incubation , the culture medium was aspirated and wells rinsed three times with incomplete medium to remove extracellular particles .
cell - associated fluorescence was measured at an excitation wavelength of 490  nm and an emission wavelength of 525  nm .
j774  cells were cultured as above in 96-well plates at a density of 0.5    10  cells / well .
solutions of amb and deta / no , or a suspension of particles in dmem without fbs or supplements were added to a final concentration of 50  m of either drug in wells assigned .
after 2  h , the wells were washed three times and complete medium was replaced .
no2 accumulation in the supernatant of cultured cells was used as an indicator of no production by the culture .
supernatants from duplicate wells were recovered sequentially at 5 , 15 , and 30  min , followed by 1 , 3 , 6 , 12 and 24  h. no in supernatant was analyzed using griess ' reagent ( 0.2% naphthylenediamine dihydrochloride , and 2% sulphanilamide in 5% phosphoric acid ) against a sodium nitrite standard curve at 540  nm ( powerwave - xs , bio - tek instruments ) .
hemolysis induced by the particles in fresh citrated mouse blood was determined ( wang et  al . , 2006 ) . with permission from the institutional animal ethics committee of csir - cdri , ( iaec/2008/25/renew 06 - 65/13 ) ,
particles were incubated with citrated blood at 37  c for 2  h , and then centrifuged at 3000  rpm for 5  min .
the positive control comprized rbc suspension in 1% triton - x solution in pbs for surfactant lysis , and the negative control was rbc in pbs .
the in - vitro cytotoxicity of formulations towards j774  cells was measured by an mtt assay .
cells were seeded in 96 well plates at a density of 0.2    10 and were allowed to adhere overnight . the medium was replaced and cells incubated for different time intervals ( 6 , 48 and 72  h ) with different formulations at equivalent doses of particles .
these doses generated a concentration of 50  m of amb or deta / no in wells exposed to particles containing single drugs and 25  m in respect of particles containing both .
cells were then washed twice and incubated in the presence of 5  mg / ml mtt for 4  h at 37  c .
dmso ( 50  l ) was added to each well to solubilize formazan crystals .
the optical density was measured using a multiwell scanning spectrophotometer ( biotek microplate reader , dynatech laboratories inc .
the strain mhom / in/1980/dd8 ( henceforth , dd8 ) strain were grown and maintained at 25  c in m199 medium supplemented 10% fbs and 1% antimycotic - antibiotic mixture .
promastigotes of late log phase were seeded at a concentration of 5    10  cells / well in a 96-well flat - bottomed micro - titre plate ( cellstar , nc , usa ) and different concentrations of amb and/or deta / no ( 12.5 , 25 and 50  g / ml of either drug ) were generated by adding free drugs or particles .
the cells were incubated for 48  h  at 25  c after treatment .
the promastigotes were counted using a haemocytometer under a light microscope at 48  h and 72  h. the % inhibition of parasite growth of treated cultures were compared to that of untreated control .
j774a.1  cells ( 10  cells / well ) were taken in 16-well chamber slides ( nunc , il , usa ) and infected with late log phase dd8 promastigotes at a multiplicity of infection of 10 .
prior to infection , the cells were washed with medium to remove the non - adherent macrophages .
after 12  h , the chamber slides were washed three times with pbs ( ph 7.2 ) to remove extracellular promastigotes and complete medium replaced .
different concentrations of drugs ( 12.5 , 25 and 50  g / ml ) were added to the wells in triplicate and incubated for 48  h. cells were fixed in absolute methanol , stained with giemsa stain and examined under oil immersion objective ( zeiss axiovert 25 ) .
untreated infected macrophages were used for comparing numbers of infected cells and amastigotes per cell .
all experiments were conducted under approval / supervision of the institutional animal ethics committee of csir - cdri ( reference number iaec/2008/25/renew 06 ( 65/13 ) ) .
the anti - leishmanial activity of particles containing amb , deta / no or both drugs was determined using fungizone and ambisome as comparators , at drug - equivalent doses of 1  mg / kg body weight .
the syrian golden hamster model was employed ( gupta et  al . , 2007 ) .
hamsters ( n  =  3 per group ) were infected with leishmania donovani dd8 promastigotes and after 30 days of established infection , the animals were injected 1  mg / kg equivalent doses of particles or comparators for 5 consecutive days by the intraperitoneal route .
splenic dab smears obtained by necropsy were microscopically examined using giemsa - stained imprints wherein the parasite burden was expressed in terms of leishman - donovan units ( ldu ) , according to the stauber 's formula ( stauber et  al . , 1958 ) .
ldu or stauber 's count  =  ( number of amastigotes per 1000 nucleated cells    weight of organ in grams )    2    10 the percent parasite suppression ( ps ) was calculated using the following formula.ps=100[1(pt/pc)]where pt and pc denote the mean stauber 's count of treated group and control group respectively .
differences between formulations were compared using one - way analysis of variance ( anova ) on ranks .
all comparisons of amb and deta / no individually were at equal concentrations , regardless of whether particles or free drug were used .
amounts of amb exposed to groups of animals or cells receiving two - drug particles were lower than those receiving amb particles or free drug , since these doses were normalized for 50  m - equivalents of deta / no . for evaluation of efficacy , the power of the study was first calculated using the primary outcome measure of reduction in spleen parasite burden from 400 to 150 amastigotes/100 macrophages after 7 days of stopping treatment .
n  =  3 was chosen to estimate differences by non - parametric analysis or by one - way anova on ranks at a power > 0.95 .
the particle size and size distribution , zeta potential , amb and/or deta / no content , and encapsulation efficiency in respect of different formulations is shown in table  1 .
the particles appeared as spheres or deformed spheres in scanning electron microscopy ( fig 1a ) and displayed gaussian size distribution in suspension ( fig 1b ) .
the in  vitro drug release data were tested against several kinetic models of drug release to elucidate the mechanism , and it was concluded that diffusion controlled release conforming to the higuchi model supervened ( costa and sousa lobo , 2001 ) . a linear relationship was observed between the amount of drug released and square root of time , with correlation coefficients ( r ) of 0.82 and 0.93 respectively for amb and 2-drug particles .
these results suggest a role of deta / no as a release modifier for amb in 2-drug particles .
particles containing traces of fitc alone ( blank particles ) were taken up as avidly as those containing amb within 2  h of exposure to j774 mouse macrophages in culture , as shown in fig 2a .
particles containing deta / no alone or in combination with amb were indistinguishable in terms of uptake , but differed from amb or blank particles in extent of fluorescence imparted to the cells .
brighter staining by blank and amb particles as compared to deta / no or two - drug particles is attributable to the polydispersity of the different preparations .
culture supernatants of j774 macrophages exposed to a solution of deta / no , or to particles containing the prodrug for 2  h were monitored for no- levels for the next 12  h , as shown in fig 2b .
no generated by soluble deta / no peaked within 15  min and declined , while particle - incorporated deta / no resulted in no levels peaking later and lower .
time curve ( auc ) of no elicited by soluble and particulate deta / no were statistically in significant ( one - way anova on ranks , p  <  0.05 ) , even when the dose of deta / no was reduced to half in respect of particles containing both deta / no and amb .
panel 3c shows results obtained when cells were infected with l.  donovani strain dd8 at moi of 10 , 6  h prior to treatment .
the control in panel 3b represents viability of uninfected cells receiving no treatment , and in panel 3c , infected cells receiving no treatment . in  vitro evaluation of cytotoxicity
was carried out over 72  h , and yielded intriguing data in respect of deta / no . it appears that deta / no protected cells from death over the first 6  h of exposure .
amb is a well - known cytotoxic agent , and reduced cell viability to 20% of untreated controls within 6  h , regardless of whether it was administered as particles or in solution .
the results were transformed to display average % inhibition of parasite survival , and are shown in fig . 
particles containing deta / no alone showed little inhibitory activity at 12.5  m and 25  m , but at higher concentrations , they led to a significant ( p  <  0.05 ) inhibition of promastigotes . at 50  m concentration , free deta /
no effected 34.9% parasite inhibition , while particle - borne deta / no had no significant difference in efficacy ( 41.71% ) .
amb in solution and amb particles showed 81.12 and 89.91 percent inhibition at 50  m respectively .
particles containing both amb and deta / no inhibited 98.51% of free - living parasites at 50  m .
activity against l.  donovani dd8 amastigotes was evaluated by staining and counting numbers of parasites / macrophage nucleus .
deta / no and amb in solution at 50  m resulted in per cent inhibition of 28.04    8.18 and 61.54    3.28 respectively with respect to infected , untreated controls .
particles containing these two agents individually resulted in 51.13    3.95 and 65.90    5.97% inhibition respectively , which was significantly greater than that observed with blank particles ( p  <  0.00001 ) .
when both agents were incorporated together in particles , inhibition was 76.41    3.58% .
two - drug particles induced significantly higher inhibition as compared to particles containing either of the two drugs alone ( p  =  0.00012 , 0.046 in respect of deta / no and amb ) .
blank or drug - free particles also inhibited promastigote survival , presumably by means of non - specific membrane disruption or stress .
ambisome and fungizone were the comparators employed here and the percent parasite suppression observed by their administration was 88.30    1.76% and 74.02    5.38% respectively ( table  2 ) .
particles containing deta / no possessed significant in  vivo leishmanicidal efficacy ( 73.10    2.50% suppression ) that was comparable to fungizone , but not ambisome. as expected , 2-drug particles displayed the highest activity ( 92.82    1.03% suppression ) among all formulations .
deta / no particles effectively reduced the splenic parasite burden at a dose of 1  mg / kg / day under the treatment regimen employed .
the significantly higher activity of 2-drug particles is attributable to the combined anti - parasitic effect of both amb and no prodrug .
there was no significant difference observed in the leishmanicidal activities between particles containing deta / no and fungizone ( p  >  0.05 ) .
0.05 ) were observed in activities of other particles and the commercial formulation , having leishmanicidal activity in the rank - order 2-drug particles  >  ambisome>amb particles  >  fungizone. the mean ranks of treatment groups compared to the untreated control group ( mean rank 2.0 ) in a kruskal 
wallis anova were 2.0 , 6.3 , 6.7 , 11.0 , 14.0 and 17.00 respectively .
the use of amb particles in plga has been proposed by several researchers over the years ( van de ven et  al . , 2012 , jain et  al . , 2014 , ribeiro et  al . , 2014 ,
, 2015 , gupta et  al . , 2015 ) . a particle formulation for targeted delivery of anti - leishmanial agents to macrophages as reported earlier ( pandya et  al . ,
. however , in view of the relatively large dose volume to be administered as a bolus , it would be advantageous if a drug combination regimen could reduce the required dose of amb ( davidson , 1991 ) .
the present results suggest that the no prodrug deta / no possesses intrinsic leishmanicidal activity against promastigotes and amastigotes , especially if incorporated in particles , comparable to the commercial formulation fungizone. it can be used alone , or preferably , in combination with amb for additive effects on parasite inhibition if formulated as particles suitable for intravenous injection .
particles reported here were prepared by a scalable process ( section 2.2 and table  1 ) , not requiring the use of ultrasonic energy .
all particles in the preparation , however , were not spherical although the preparations were fairly monodisperse ( fig . 
matrix - controlled release of amb was observed from the particles , suggesting that intracellular concentrations of released drug would follow similar temporal profiles when the particles are taken up by macrophages .
we did not study the release of the prodrug ( deta / no ) in these experiments , preferring instead to study the time kinetics of the active moiety ( no ) in culture supernatants . as evident from fig . 
2b , no levels were sustained at 2030  m for a period of 24  h when mouse macrophages were exposed to particles containing deta / no . these concentrations are similar to values reported in respect of macrophages recovered from mice receiving protective immunization against leishmania infantum ( khoshgoo et  al . , 2008 ) .
it is premature to speculate whether cell death induced by amb and/or deta / no would provide benefit in chemotherapy of leishmaniasis by virtue of denying intracellular 
4 , however , support the view that host cell death could affect inhibition of parasite growth .
the findings are encouraging with regard to the safety of proposed formulation , but more detailed investigations are required for a tenable claim of safety .
it has been demonstrated elsewhere that lactide - glycolide particles show leishmanicidal activity ( costa lima et  al . , 2014 ) .
there was significant enhancement of activity of deta / no when incorporated in particles , towards amastigotes .
while the differences in the extent of parasite inhibition by soluble versus particle - borne amb are not statistically significant , it is expected that lower concentrations might reveal clearer differences in efficacy at equivalent dose .
further , the lack of difference between activities of particulate and soluble amb suggests that the drug is capable of sufficient intracellular accumulation in  vitro , without the need for targeting .
however , it is likely that uptake of particles may induce macrophages to mount parasiticidal innate responses , especially if the contents release no , a known mediator of such responses ( verma et  al . , 2012 ) .
analogous results from our work on macrophages infected with an unrelated pathogen , m. tuberculosis , suggest that the presence of particles in the macrophage 's intracellular compartment induces a variety of processes indicative of classical activation ( sharma et  al . , 2007 ) .
both amb and deta / no can reportedly elicit the expression of inos in macrophages ( larabi et  al . , 2001 )
, presumably leading to enhanced intracellular no . it is also possible that no might act directly on the parasite membrane .
we submit that these particles might be of interest to researchers investigating host - pathogen dialectics in the context of leishmania where intracellular generation of no could result in important outcomes .
the deta / no particles reported here can serve as a biochemical tool to investigate effects of exogenous supplementation of what is essentially an innate host response .
we found compelling evidence of pre - clinical superiority of the two - drug particles in comparison to marketed liposomal formulations of amb , and non - inferiority of particles containing deta / no alone ( table  2 ) .
thus , we submit that the prodrug - in - particle approach to passively target no to macrophages deserve further investigation , especially to establish appropriate dosing regimens . finally , we submit that the only way to clinically deploy the invaluable contributions of no to stimulation of host macrophage responses as well as parasiticidal chemotherapy of leishmania infection is through targeted delivery of no donors to the macrophage cytosol .
 we investigated efficacy of nitric oxide ( no ) against leishmania donovani . 
 no is a mediator of host response to infection , with direct parasiticidal activity in addition to its role in signalling to evoke innate macrophage responses . 
 however , it is short - lived and volatile , and is therefore difficult to introduce into infected cells and maintain inracellular concentrations for meaningful periods of time . 
 we incorporated diethylenetriamine no adduct ( deta / no ) , a prodrug , into poly(lactide - co - glycolide ) particles of 200  nm , with or without amphotericin b ( amb ) . 
 these particles sustained no levels in mouse macrophage culture supernatants , generating an area under curve ( auc0.08 - 24h ) of 591.2    95.1  mm    h. free deta / no resulted in no peaking at 3  h and declining rapidly to yield an auc of 462.5    193.4 . particles containing amb and deta / no were able to kill 98% of promastigotes and 76% of amastigotes in 12  h when tested in  vitro . 
 promastigotes and amastigotes were killed less efficiently by particles containing a single drug either deta / no ( 42% , 35% ) or amb ( 90% , 50% ) alone , or by equivalent concentrations of drugs in solution . in a pre - clinical efficacy study of power > 0.95 in the hamster model , deta / no particles were non - inferior to fungizone but not ambisome , resulting in significant ( 73% ) reduction in spleen parasites in 7 days . particles containing both deta / no and amb were superior ( 93% reduction ) to ambisome. we conclude that no delivered to the cytosol of macrophages infected with leishmania possesses intrinsic activity and adds significantly to the efficacy of amb . 
Introduction
Material and methods
Results
Discussion
Conflicts of interest