Amphotericin B (AmB) was formulated in tripalmitin based nanosize lipid particles (emulsomes) stabilized by egg phosphatidylcholine (PC) for macrophage targeting for the treatment of visceral leishmaniasis (VL). Emulsomes were prepared by cast film technique followed by sonication to obtain particles of nanometric size range and further modified by anchoring them with macrophage-specific ligand (p-aminophenyl-α-D-mannoside, PAM). The surface modified emulsomes and their plain counterparts were characterized for size, shape, entrapment efficiency, zeta-potential, in-vitro drug release and ligand binding specificity. The antileishmanial activity of AmB-deoxycholate (AmB-Doc) and emulsome entrapped AmB was tested in vitro in Leishmania donovani infected macrophage-amastigote system (J774A.1 cells), which showed higher efficacy of PAM-anchored AmB emulsomes (TPEs-PAM) over plain AmB emulsomes (TPEs) and AmB-Doc. The in vivo antileishmanial activity of the AmB (0.5 and 1mg/kg respectively) was tested against VL in L. donovani infected hamsters in which formulation TPEs-PAM eliminated intracellular amastigotes of L. donovani within splenic macrophages more efficiently (52.44 ± 3.9 % and 68.03 ± 4.2 % parasite inhibition, PI) than the formulation TPEs (32.86 ± 2.4 % and 39.66 ± 3.8 % PI) and AmB-Doc (20.98 ± 2.10 % and 24.43 ± 3.55 % PI). We concluded that PAM-anchored emulsomes could fuse with the macrophages of liver and spleen due to ligand-receptor interaction and could target the bioactives inside them. Our results suggest that these newer formulations (plain and ligand-anchored emulsomes) are a promising alternative to the conventional AmB-Doc formulation for the treatment of VL.Abstract Amphotericin B (AmB) was formulated in tripalmitin based nanosize lipid particles (emulsomes) stabilized by egg phosphatidylcholine (PC) for macrophage targeting for the treatment of visceral leishmaniasis (VL). Emulsomes were prepared by cast film technique followed by sonication to obtain particles of nanometric size range and further modified by anchoring them with macrophage-specific ligand (p-aminophenyl-α-D-mannoside, PAM). The surface modified emulsomes and their plain counterparts were characterized for size, shape, entrapment efficiency, zeta-potential, in-vitro drug release and ligand binding specificity. The antileishmanial activity of AmB-deoxycholate (AmB-Doc) and emulsome entrapped AmB was tested in vitro in Leishmania donovani infected macrophage-amastigote system (J774A.1 cells), which showed higher efficacy of PAM-anchored AmB emulsomes (TPEs-PAM) over plain AmB emulsomes (TPEs) and AmB-Doc. The in vivo antileishmanial activity of the AmB (0.5 and 1mg/kg respectively) was tested against VL in L. donovani infected hamsters in which formulation TPEs-PAM eliminated intracellular amastigotes of L. donovani within splenic macrophages more efficiently (52.44 ± 3.9 % and 68.03 ± 4.2 % parasite inhibition, PI) than the formulation TPEs (32.86 ± 2.4 % and 39.66 ± 3.8 % PI) and AmB-Doc (20.98 ± 2.10 % and 24.43 ± 3.55 % PI). We concluded that PAM-anchored emulsomes could fuse with the macrophages of liver and spleen due to ligand-receptor interaction and could target the bioactives inside them. Our results suggest that these newer formulations (plain and ligand-anchored emulsomes) are a promising alternative to the conventional AmB-Doc formulation for the treatment of VLAmphotericin B (AmB) was formulated in tripalmitin based nanosize lipid particles (emulsomes) stabilized by egg phosphatidylcholine (PC) for macrophage targeting for the treatment of visceral leishmaniasis (VL). Emulsomes were prepared by cast film technique followed by sonication to obtain particles of nanometric size range and further modified by anchoring them with macrophage-specific ligand (p-aminophenyl-α-D-mannoside, PAM). The surface modified emulsomes and their plain counterparts were characterized for size, shape, entrapment efficiency, zeta-potential, in-vitro drug release and ligand binding specificity. The antileishmanial activity of AmB-deoxycholate (AmB-Doc) and emulsome entrapped AmB was tested in vitro in Leishmania donovani infected macrophage-amastigote system (J774A.1 cells), which showed higher efficacy of PAM-anchored AmB emulsomes (TPEs-PAM) over plain AmB emulsomes (TPEs) and AmB-Doc. The in vivo antileishmanial activity of the AmB (0.5 and 1mg/kg respectively) was tested against VL in L. donovani infected hamsters in which formulation TPEs-PAM eliminated intracellular amastigotes of L. donovani within splenic macrophages more efficiently (52.44 ± 3.9 % and 68.03 ± 4.2 % parasite inhibition, PI) than the formulation TPEs (32.86 ± 2.4 % and 39.66 ± 3.8 % PI) and AmB-Doc (20.98 ± 2.10 % and 24.43 ± 3.55 % PI). We concluded that PAM-anchored emulsomes could fuse with the macrophages of liver and spleen due to ligand-receptor interaction and could target the bioactives inside them. Our results suggest that these newer formulations (plain and ligand-anchored emulsomes) are a promising alternative to the conventional AmB-Doc formulation for the treatment of VL.

• Amphotericin B

• Targeting

• Macrophage

• Emulsome

• Visceral leishmaniasis

Kumar N, Sharma P, Jaiswal A, Dube A, Gupta S (2016) Development and Evaluation of P-Aminophenylmannopyranoside Anchored Emulsomes for Treatment of Experimental Visceral Leishmaniasis. Ann Clin Cytol Pathol 2(6): 1042.

VL: Visceral leishmaniasis; AmB: Amphotericin B; PAM: paminophenylmannopyranoside; PE: Phosphatidylethanolamine; Con-A: Concanavalin-A

Visceral leishmaniasis (VL) results from infection of the macrophages of the liver, spleen and bone marrow with protozoal parasite Leishmania donovani. India alone may contribute as much as 40–50% of these with 90% occurrence in the state of Bihar [1,2]. WHO model list of essential medicines 14th edition (March 2005) enlists only antimonials for the treatment for VL, but there is emerging evidence that the rates of response to the antimonials are declining due to the appearance of resistance, and relapses are common [3]. Amphotericin B (AmB) provides substantial leishmanicidal activity as well, and its use results in fewer treatment failures and relapses. However, the important side effects, mainly nephrotoxicity, produced by this drug at therapeutic doses have often led to its refusal as a first-choice treatment [4].

For diseases of microbial etiology, the intracellular localization of the pathogens necessitates the administration of relatively high doses of the cytotoxic drugs for the effective killing of the pathogens, thereby causing the side effects [5]. One such approach to increase the efficacy and to reduce the dose related toxicity of these agents is to target the drug molecule to the phagolysosomes of the MPS where the leishmania parasites reside [4,6]. Thus, there is an urgent need to develop safe drugdelivery strategies for existing molecules. Novel drug delivery systems, such as liposomes, microspheres and nanospheres can result in higher concentrations of AmB in the liver and spleen but lower concentrations in the kidney and lungs, thus decreasing the toxicity of AmB. The ability of nanocarriers to be taken up by MPS makes them ideal vehicle for selective transport of drug to target tissues in disease where phagocytic cells are involved.

However, most of these colloidal carriers fail to deliver the drug to diseased area efficiently due to nonspecific MPS uptake and poor target specificity. The natural passive uptake suffers from many inherent disadvantages like poor drainage at the site of injection and nonhomogeneous distribution to various macrophages-specific tissues.

Surface modification of these carriers with site-specific ligands further facilitates their rate and extent of macrophage accumulation of drug which may further reduce the dose size and dose frequency of already licensed lipid AmB formulations for the treatment of VL [7]. The exclusive presence of mannose/fucose receptors on macrophages has been exploited for developing an efficient macrophages-directed drug carrier [8,9].

AmB is a potent polyene antibiotic, available as a micellar solution (Fungizone®), liposome (AmBisome®), AmB lipid complex (Abelcet®), and AmB colloidal Dispersion (Amphocil®) for effective treatment of VL and some other systemic fungal infections like candidiasis. However, the utility of these new products is greatly limited due to their high costs. So there is a need for the development of low cost formulations [10].

The objective of the proposed work is the effective and sitespecific localization of AmB inside the macrophages to treat the VL using emulsomes. Emulsomes are a new generation colloidal carrier system in which internal core is made of fats and triglycerides which is stabilized by high concentration of lecithin in the form of o/w emulsion. By virtue of solidified or semisolidified internal oily core it provides a better opportunity to load lipophilic drugs like AmB in high concentration, simultaneously a protracted controlled release can also be expected. Also, due to their colloidal nature, they can be passively taken up from the blood stream by the macrophages of the liver and spleen (host for the leishmania parasite) after intravenous or intracardiac administration [11] and hence can be very useful for the treatment of VL. The composition and manufacturing procedures of the emulsomes make feasible the production of a stable final product that could be an economically interesting alternative to the current commercial lipid AmB formulations.

In our previous work, we have developed mannosylated emulsomes and solid lipid nanoparticles for macrophage targeting using AmB against VL [10,11]. In the present study, we have developed emulsomes using different lipid and coated them with another macrophage specific ligand, p-aminophenylmannopyranoside (PAM) to check its antileishmanial efficacy and the results of free AmB, plain and ligand-anchored emulsomes entrapped AmB are compared.

Drugs and chemicals

Amphotericin B was a kind gift from Life care Innovations Pvt. Ltd., Gurgaon, India. AmB-deoxycholate (AmB-Doc, AmphotretTM) was purchased from Bharat Serums and Vaccines Limited., Ambernath, India. Egg phosphatidylcholine (PC), phosphatidylethanolamine (PE), cholesterol, p-aminophenylmannopyranoside (PAM), concanavalin A (Con-A) and Sephadex G-50, Dialysis tubing (Mw 12 kDa) were purchased from Sigma Chemicals Company (St Louis, MO, USA). Tripalmitin, glutaraldehyde and trichloroacetic acid were purchased from Himedia Laboratories Pvt Ltd., Mumbai. Potassium dihydrogen phosphate, disodium hydrogen phosphate, and mannitol were purchased from Rankem Laboratory Reagents, New Delhi. Chloroform and all other chemicals were of pure analytical grade and used as procured.

Parasite

The WHO reference strain, L. donovani (MHOM/IN/80/Dd8) promastigotes were cultured in RPMI-1640 medium (Sigma, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma, USA), penicillin (100 U/ml) and streptomycin (100µg/ml) at 268C. Parasites were also maintained through in vivo serial passage (amastigote to amastigote) in hamsters [12]. Animal host Laboratory bred male Syrian golden hamsters (Mesocricetus auratus, 45–50 g) from animal house facility of Central Drug Research Institute (CDRI) were used as the experimental host. They were housed in plastic cages in climatically controlled rooms and fed with standard rodent food pellet (Lipton India Ltd, Bombay) and water ab libitum.

Preparation of emulsomes

Emulsomes containing AmB were prepared following the already established and reported method of our laboratory [11]. To a 250 ml round-bottomed flask, AmB was dissolved in methanol by sonication. In a separate beaker PC, tripalmitin and cholesterol were codissolved in chloroform. Both organic solutions were mixed, and the organic solvent was evaporated until complete dryness under reduced pressure using a rotary flash evaporator in to form a thin lipid film on the walls of the round-bottom flask. The dried film was hydrated with phosphate-buffered saline (PBS) pH 7.4 (4 ml) and homogenized by ultrasonication at 50% amplitude to obtain emulsomes of nanometric size range. The free un-entrapped drug was removed by passing the dispersion through a sephadex G-50 column [13].

Optimization of emulsomes

Emulsomes were optimized for AmB to lipid ratio, PC to tripalmitin ratio and sonication time. For optimization of AmB to lipid ratio, PC to tripalmitin ratio (1:1 w/w), cholesterol (4% w/w of PC) and sonication time (4 min) were kept constant while AmB content was varied at different weight percent ratio levels, i.e. (1, 2, 4, 6, 8% weight of the lipid) in different formulations (Table 1) for determining optimum AmB content. Average particle size of different formulations was determined by particle size analyzer (Delsa NanoC, Beckman Coulter Pvt. Ltd., USA) and percent drug entrapment in different formulations was also determined as described previously [16]. Emulsomes with optimum AmB to lipid ratio were optimized for optimum sonication time in terms of average particle size. PC to tripalmitin ratio (1:1w/w), cholesterol (4% w/w of PC) and AmB to lipid ratio (optimized) were kept constant while sonication time was varied (i.e. 0, 2, 4, 6, 8 min) for different formulations. Emulsomes with optimum AmB to lipid ratio and sonication time were optimized for optimum PC to trilaurin ratio in terms of percent drug entrapment and toxicity towards erythrocytes. The emulsomes with fixed cholesterol (4% w/w of PC) and different PC to tripalmitin ratios (0.6:1, 0.8:1, 1:1, 1.2:1, 1.4:1 w/ ratios) were prepared. AmB content and sonication time however were kept constant at its optimum level. Emulsomes were evaluated for percent drug entrapment and toxicity to mammalian cells in terms of percent haemolysis.

Percent haemolysis was determined by the method reported in literature [7]. Percent haemolysis and percent drug entrapment were plotted against PC to tripalmitin ratio, from which optimum PC to tripalmitin ratio was determined.

Preparation of p-aminophenyl-mannopyranoside (PAM)-anchored emulsomes

For the preparation of PAM- anchored emulsomes, firstly phosphatidylethanolamine (PE)-containing emulsomes were prepared by incorporation of PE as one of the phospholipids at 10% w/w of PC using optimized formulation of emulsomes [11]. Now, mannosylated ligand (PAM) was inserted into the lipid bilayer using the PE end groups (Figure 1). PAM was linked to PE -containing emulsomes by the method described earlier [14,15]. A 2.5 ml of PE-emulsome dispersion (~30 mg lipids) in PBS (7.4) was mixed with 20.0 mg PAM contained in 2.0 ml of 0.9% w/v aqueous NaCl solution. Glutaraldehyde (0.5 ml of 25% v/v aqueous solution) was then slowly added to the dispersion to a concentration level 3 mM and the mixture was incubated for 5 min at 20°C. The -NH2 group of PE-emulsomes was coupled with p-aminophenyl-mannopyranoside using glutaraldehyde as coupling agent [15]. Uncoupled glycosides (sugar derivatives) and glutaraldehyde were removed through dialysis using dialysis membrane (12 kDa) technique against the same buffer (Figure 1)

In-vitro characterization

Developed formulations were characterized before and after surface ligand anchoring. Prepared emulsomes were evaluated for their shape by both transmission electron microscopy (TEM) (Hitachi, Japan) and scanning electron microscopy (SEM) (JEOL, EVO-50 Japan). A drop of the sample was placed on to a carbon coated copper grid to leave a thin film on the grid. Phosphotungstic acid (1%) was used as a negative stain. A drop of the staining solution was added on to the film and the excess of the solution was drained off. The grid was allowed to dry at ambient temperature and subjected to TEM analysis. Size, polydispersity index (PDI) and zeta-potential was determined by photon correlation spectroscopy method using Bechman Coulter Zetasizer (NanoDelsaC; Bechman Coulter Pvt. Ltd., UK).

Percent drug entrapment was determined and expressed as the ratio of experimentally measured amount of drug in dispersion and initial amount used for entrapment. Centrifugation was done at 3000 rpm for 3 min to remove unentrapped drug. Vesicles (free of unentrapped drug) were lysed by adding 1.0 ml of 0.1% v/v triton X-100 and liberated contents were analyzed for AmB content spectrophotometrically at 405 nm [5].

In-vitro drug release

The in-vitro drug release profiles of AmB from different emulsomal formulations were determined using a dialysis tube (MWCO 12 kDa; Sigma Chemical Co., USA) method. A 2 mL volume of the formulation was taken in the dialysis tube (donor compartment) and placed in a beaker containing 100 ml of PBS/ DMSO (95%:5% v/v) (receptor compartment). The assembly was placed over a magnetic stirrer at 100 rpm and the temperature was maintained at 37 ± 1°C throughout the study. Samples (5 ml) were withdrawn periodically and after each sample withdrawal the medium was compensated immediately with fresh dialyzing medium PBS/DMSO (95%:5% v/v) while maintaining strict sink conditions. The samples were analyzed for AmB content spectrophotometrically at 405 nm [4].

In vitro ligand binding specificity

In vitro ligand-specific activity was performed to assess the surface orientation and availability of accessible mannose residues of PAM after the formation of PAM-anchored emulsomes using concanavalin-A (Con-A) lectin as reported with slight modification [16]. The affinity towards exogenously provided lectin Con-A was used as a measure of activity of PAM-anchored emulsomes. A 200µL sample of the original emulsomal dispersion (both plain and PAM-anchored) was diluted 10-times with PBS (pH 7.4) and 1mL Con-A (1mg/ml) in PBS (pH 7.4) with 5mM CaCl2 , and 5mM MgCl2 was added to it. The increase in turbidity at 550 nm was monitored spectrophotometrically for 2 h.

In-vitro activity against intracellular amastigotes in macrophages

Mouse macrophage adherent cell line J774A.1 was maintained in RPMI-1640 medium (Sigma, USA) supplemented with 10% heat inactivated fetal bovine serum (HIFBS) at 37?C in humidified atmosphere of 5% CO . Macrophages (105 cells/well) in 16-well chamber slides (Nunc, IL, USA) were infected with promastigotes (L. donovani, Dd8) at multiplicity of infection of 10:1 (parasites/ macrophage) and incubated at 37?C in 5%

CO2 for 12 h after which the chamber slides were washed thrice with PBS (pH 7.2) to remove non-phagocytosed promastigotes and finally supplemented with complete medium [17]. Different concentrations (0.08 and 0.16µg/ml) of 100 μl of free AmB (AmB- Doc, Mycolâ), optimized tripalmitin emulsome formulations (TPES4P4), and PAM-anchored emulsome formulations (PAM- TPEs) or (TPES4P4-PAM) in RPMI-1640 medium were added to wells in triplicate. Similarly empty TPES4P4 and empty TPES4P4- PAM without AmB but having same composition in RPMI-1640 medium were added to wells in triplicate. The untreated infected macrophages were used as control. Formulations were then removed by washing after 3 h and macrophages were placed in medium for an additional 20 h, after that they were examined for intracellular amastigotes under oil immersion objective of light microscope after methanol fixing and Giemsa staining of the slides. At least 100 macrophage nuclei were counted per well for calculating percentage infected macrophages and number of amastigotes per 100 macrophages. Percent parasite inhibition in treated wells was calculated using the following formula [10,17].