RESEARCH ARTICLE

Antileishmanial activity, pharmacokinetics and tissue distributionstudies of mannose-grafted amphotericin B lipid nanospheres

PRABHAKAR REDDY VEERAREDDY1, VENKATESWARLU VOBALABOINA1, &

NAHID ALI2

1University College of Pharmaceutical Sciences, Kakatiya University, Warangal 506009, India, and 2Indian Institute of

Chemical Biology, Jadavpur, Kolkata 700032, West Bengal, India

(Received 27 July 2008; accepted 4 October 2008 )

AbstractLeishmania parasite resides mainly in the liver and the spleen and multiplies. Effective therapy of leishmaniasis could beachieved by delivering antileishmanial drugs to these sites. Present investigations were aimed at developing lipid nanospheresof amphotericin B (LN-A) anchored with mannose to achieve targeted delivery to the liver. Mannose is specifically involved inthe recognition of parasite or appropriate ligands on the macrophage surface LN-A, and mannose-anchored lipid nanospheres(LN-A-MAN) were prepared by homogenization followed by ultrasonication method. Particle size and zeta potential weremeasured using Malvern Zetasizer. The average particle size after sterilization of LN-A and LN-A-MAN ranged from193.4 ^ 1.1 to 775.8 ^ 9.1. Leishmaniasis was induced in BALB/c mice by injecting Leishmania donovani parasitesintravenously. Infected mice were administered with a single dose (5 mg/kg body weight) of LN-A, LN-A-MAN, andFungizone (marketed product).The efficacy of the formulations was evaluated by measuring the reduction in parasite burden.Fungizone reduced 82 and 69%, LN-A reduced 90 and 85%, LN-A-MAN reduced 95 and 94% of parasite burden in the liverand the spleen, respectively. LN-A and LN-A-MAN-treated mice did not show any elevation in serum glutamate pyruvatetransaminase (SGPT), alkaline phosphatase (ALP), urea, and creatinine levels as compared with Fungizone. Pharmacokineticparameters were estimated and the concentration of amphotericin B (AmB) in mice plasma declined biexponentially and AmBconcentrations were significantly higher for LN-A- and LN-A-MAN than Fungizone-treated mice (P , 0.05). Tissuedistribution patterns were studied in different tissues such as the liver, the spleen, the kidney, and the brain of BALB/c mice.LN-A-MAN was found to distribute more rapidly to the liver and the spleen explaining the reason for higher antileishmanialactivity.

Keywords: Lipid nanospheres, amphotericin B, mannose, pharmacokinetics and tissue distribution

Introduction

Leishmaniasis is a complex of disease syndromes, with

a spectrum that has been divided into visceral,

cutaneous, and mucocutaneous forms, caused by

protozoan parasites of the genus Leishmania.

The disease is endemic in many tropical and

subtropical regions of the world, with 500,000 people

at risk (Boelaert et al. 2000). There is an estimated

prevalence of 23 million (WHO, 2002) with an

incidence of 1.0–1.5 million cases per annum of the

potentially fatal visceral leishmaniasis (VL) (Ashford

et al. 1992). VL has also emerged as an important

opportunistic infection in immunocompromised

patients, in particular those with HIV, in some regions

of the world, most notably the Mediterranean

countries (Alvar et al. 1997; Gradoni et al. 1995).

Leishmaniasis occurs from tropical to Mediterranean

regions, where the parasite is transmitted by female

sandflies of the genus Phlebotomus in the Old World

and Lutzomyia in the New World (Senior et al. 1991).

In the insect gut as well as tissue culture media, the

parasite exists as extracellular, elongated, flagellated

promastigotes. Promastigotes are injected into

the skin during a blood meal and rapidly taken

ISSN 1061-186X print/ISSN 1029-2330 online q 2009 Informa UK Ltd.

DOI: 10.1080/10611860802528833

Correspondence: V. Reddy, University College of Pharmaceutical Sciences, Kakatiya University, Warangal 506009, India. Tel: +9 187 02446259. Fax: +9 187 024 38844. E-mail: vpreddyindia@yahoo.com

Journal of Drug Targeting, February 2009; 17(2):140–147

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up by mononuclear phagocytes, where they reside in

the parasitophorous vacuole.

Amphotericin B (AmB) is a member of the polyene

class of antifungal agents. AmB, a lipophilic antibiotic

is first isolated from the soil actinomycete, Strepto-

myces nodosus, in 1953. Because of its poor oral

absorption and relative water insolubility, it is typically

supplied as an intravenous preparation (Fungizone;

Sarabhai Chemicals, Ahmedabad, India) that is

combined with the bile salt, sodium deoxycholate,

that acts as a detergent. On reconstitution with sterile

water and final preparation in 5% dextrose in water,

stable micellar dispersion is formed.

Amphotericin B exerts its antifungal activity by

irreversibly binding to ergosterol, a sterol present in

the fungal cell membrane (Warnock, 1991). This

results in a poorly functioning membrane that permits

cellular components to leak out, causing cell death.

The drug may exert either fungicidal or fungistatic

activity, depending on its concentration at the site of

infection and sensitivity of the organism (Gallis et al.

1990). Nanoform of AmB deoxycholate has high

efficacy than conventional AmB deoxycholate for the

treatment of VL (Manandhar et al. 2008). The aim of

the present study was to compare the antileishmanial

activity, pharmacokinetics, and tissue distribution of

mannose-grafted AmB lipid nanospheres with that of

Fungizone.

Experimental

Materials

Amphotericin B, Sarabhai Chemicals Limited (Vado-

dara, Gujarat India); Egg lecithin, Sigma Chemicals

(St Louis, MO, USA) (PC: 60%); soybean oil, Cargil

Foods (Gurgaon, Haryana, India); cholesterol, Quali-

gens Fine Chemicals (Mumbai, Maharashtra, India)

and Fungizone, Sarabhai chemicals.

Preparation of lipid nanospheres of amphotericin B

General method of preparation of lipid nanospheres of

amphotericin B. Amphotericin B, egg lecithin, and

cholesterol were dissolved in soybean oil, heated to

708C on a water bath, and stirred until the system is

clear. Glycerol, sucrose, and sodium oleate were

dissolved in sufficient amount of distilled water and

the aqueous phase was added to the oil phase at the

same temperature (708C). A coarse emulsion was

prepared by homogenization (Remi homogenizer) at

6000 rpm. In order to get the particle size below 1mm,

coarse emulsion formed by homogenization at

6000 rpm for 3 min was subjected to ultrasonication

using Labsonic-L (B.Braun Biotech International

GmbH, Germany) probe type ultrasonicator with

12T probe at 100 W. The blank lipid nanosphere

formulation (LN-B) was prepared in a similar manner

without drug. The formulations were filled into vials,

sealed and sterilized by autoclaving at 1218C.

Preparation of mannose-grafted lipid nanospheres of

amphotericin B (LN-A-MAN). Mannose-grafted lipid

nanospheres were prepared using para-aminophenyl-

a-D-mannopyranoside as per the reported method

(Labanyamoy Kole et al. 1999). Phosphatidyl-

ethanolamine lipid nanospheres (2.5 ml) were mixed

with 10 mg (dissolved in 2 ml PBS) of p-amino phenyl-

a-D-mannopyranoside.

Glutaraldehyde (1 ml) was added slowly to the lipid

nanospheres, and the mixture was incubated for 5 min

at 208C. Uncoupled sugar and glutaraldehyde were

removed by dialysis (Molecular weight cutoff of

20,000 kDa) for 24 h against 25 mM sodium phos-

phate buffer, pH 7.2 containing 150 mM sodium

chloride. Degree of mannosylation in liposome surface

was examined by agglutination test using sugar-

specific lectins (Surolia et al. 1975).

Preparation of amphotericin B solution. Amphotericin B

deoxycholate commercial formulation, Fungizone

(Sarabhai Chemicals, India), contains 50 mg of AmB

deoxycholate. Stock solution of 1 mg/ml of AmB was

prepared from the commercial formulation by diluting

suitably with phosphate buffer saline of pH 7.4.

Physicochemical characterization of amphotericin B lipid

nanospheres

Measurement of size. All the formulations were suitably

diluted with their aqueous phase and the particle size

was determined using Malvern Zetasizer.

Measurement of zeta potential. The formulations were

diluted with their respective aqueous phases and the

zeta potential was determined using Malvern

Zetasizer.

Entrapment efficiency and assay. The AmB drug in the

aqueous phase of the lipid nanosphere formulation

was estimated by the ultrafiltration method (Dipali

et al. 1996). Centrisort, an ultrafiltration unit, consists

of a sample recovery chamber and support base tube

was used. Filter membrane is (Molecular weight

cutoff of 20,000 Dalton) present at the base of the

sample recovery chamber. A 500ml undiluted sample

was placed in the support base and the sample

recovery chamber placed on top of the sample.

The unit was centrifuged at 3500 rpm for 15 min.

The lipid nanospheres along with encapsulated drug

remained in the support base and aqueous phase

moved into the top sample recovery chamber through

Amphotericin B lipid nanospheres 141

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membrane. Unsterilized and sterilized samples were

subjected for ultrafiltration. The amount of the drug

in the samples was estimated by HPLC. All the

formulations were subjected to assay. About 100ml of

the sample was dissolved in chloroform:methanol

mixture (1: 1) and then further dilutions were made

with mobile phase. The diluted samples were injected

into the HPLC and the total amount of AmB in the

formulations was calculated.

Assessment of antileishmanial activity of formulations

in post-infected mice. BALB/c mice (4–6 weeks old) were

each infected intravenously with 2.5 £ 107 amastigotes.

The mice were maintained for 60 days (Tuhina et al.

2000). A single dose (5 mg/kg) of Fungizone or

formulations lipid nanospheres of amphotericin B

(LN-A) and mannose-anchored lipid nanospheres

(LN-A-MAN) were injected intravenously on day 61

and treated as day 1 of treatment. Six mice from each

group were killed 15 days after injection and parasite

load was determined by the weights and microscopic

examination of Geimsa-stained impression smears of

the liver and the spleen. The parasite load was expressed

as Leishman Donovan Units (Stauber et al. 1958).

Toxicity studies. Toxicity studies were conducted in

normal BALB/c mice to know whether the

entrapment of AmB in LN is safe or not. Fungizone,

LN-A, and LN-A-MAN were injected intravenously

(equivalent to 5 mg AmB/kg body weight). Serum

levels of enzymes (serum glutamate pyruvate

transaminase (SGPT) and alkaline phosphatase

(ALP)) and biochemical markers (creatinine and

urea) were estimated on day 15 following

administration. The kits are purchased from CDR

Diagnostics (Hyderabad, India).

Pharmacokinetics and tissue distribution studies

Animals. BALB/c mice weighing between 20 and 25 g

were used for pharmacokinetics and tissue

distribution studies. The animals were procured

from National Institute of Nutrition, Hyderabad

(NIN). All animal experiments were evaluated and

approved by the animal and ethics review committee

of Faculty of Pharmaceutical Sciences, Kakatiya

University, Warangal, India. The animals were

acclimatized for at least one week before

experimentation, fed with standard NIN diet and

allowed tap water ad libitum.

Pharmacokinetics and tissue distribution of formulations

of amphotericin B in mice. BALB/c mice (weighing

20–25 g) were divided into groups. LN-A and LN-A-

MAN, AmB deoxycholate commercial formulation

(Fungizonee) were administered at a dose of 5 mg/kg

via tail vein. At predetermined time points (5, 10, 15,

and 30 min and 1, 2, 4, 6, 8, 16, and 24 h), animals (3)

were euthanized by cervical dislocation and dissected.

Tissues of interest (the liver, the spleen, the kidney,

and the brain) and blood were collected.

Statistical analysis. The data were statistically

processed by ANOVA (GraphPad, El Camino Real,

CA, USA). Differences yielded a value of P , 0.05

that were considered statistically significant.

Results

Formulation of mannose-grafted lipid nanospheres

of amphotericin B

The components, size, and surface charge of the

nanospheres influence their biodistribution (Tuhina

et al. 2000). In addition, specific ligands anchored on

the surface of these nanospheres leads to the targeting

to specific sites. Mannose is linked to phosphatidy-

lethanolamine by in situ reaction to anchor mannose

on lipid nanospheres.

It is possible to target the delivery of drugs to

specific sites using homing devices on the surface of

colloidal particles. The mannose-grafted pentamidine

liposomes were best in lowering the spleen leishmanial

parasite load in comparison with those bearing glucose

or galactose (Banerjee et al. 1996).

Measurement of size

Size is the most prominent feature of nanospheres that

influences biodistribution.

Average size and polydispersity index of different

formulations of AmB were measured by Malvern

Zetasizer, which is based on the principle of photon

correlation spectroscopy at a fixed angle of 908 and at a

temperature of 258C. Each value reported is the

average of the three measurements. The polydispersity

index measures the size distribution of the nanopar-

ticle population.

The average particle size after sterilization of LN-A

and LN-A-MAN ranged from 193.4 ^ 1.1 to

775.8 ^ 9.1, respectively. The small size obtained

with AmB may be attributed to its amphiphilic nature.

LN-A-MAN formulations have shown larger size due

to specific arrangement of mannose around the lipid

nanospheres when compared with normal LN-A.

Entrapment efficiency and assay

A linearity exists between the concentration of AmB

injected and peak area over the concentration range of

1–6mg/ml. Samples obtained after ultracentrifugation

were analyzed by validated HPLC method (Echevarria

P. R. VeeraReddy et al.142

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et al. 2000). Entrapment efficiency was calculated to

find out the amount of AmB present in lipid

nanospheres. The percent entrapment efficiency data

are presented in Table 1.

Assessment of antileishmanial activity of formulations

in post-infected mice

The results show that AmB entrapped in lipid nano-

spheres (a single dose of 5 mg AmB/kg) could signifi-

cantly reduce the liver and the spleen parasite burden

indicating efficient localization in the liver and the

spleen. Fungizone, LN-A, and LN-A- MAN reduced

the parasite burden in the liver and the spleen to 82 and

69%, 90 and 85%, and 95 and 94%, respectively, after

15 days post infection. The potential of using LN as a

delivery system is strengthened by the fact that both LN

and the leishmanial parasites are taken up by the same

reticuloendothelial cells, creating an ideal situation for

studying drug–parasite interaction. Mannose-grafted

nanospheres are used to target the specific sites.

Mannose and glucose are specifically involved in the

recognition of the parasites or appropriate ligands on the

macrophage surface (Banerjee et al. 1996).

Toxicity studies

To carryout toxicity studies of all the formulations,

5 mg/kg body weight of AmB was used. The normal

level of SGPT and ALP are in the range of

5–35 units/ml and 3–13 KA units/dl, respectively,

while the creatinine and urea plasma levels are up to

20 and 250 mg/dl, respectively. Present investigations

estimated the specific levels of enzymes, such as SGPT

and ALP, that are related to normal liver functions on

day 15 after injection with formulations and pure AmB

(5 mg/kg body weight) compared to LN-A-treated

mice. LN-A-treated mice did not show any elevation in

their SGPTand ALP levels. In order check the effect of

AmB on renal function, urea and creatinine levels were

also assayed. With Fungizone (5 mg/kg body weight),

very high levels of these biochemical markers of renal

function are observed whereas the LN formulations did

not affect the kidneys significantly.

Toxicity studies were conducted to know whether the

entrapment of AmB in LN is safe or not. The enzyme

levels ALP and SGPT in the serum of BALB/c mice

treated with LN-A and other formulations were normal

indicating normal functioning of the liver. LN of AmB

entrapped in LN has not shown any nephrotoxicity.

Pharmacokinetics of formulations of AmB in mice

The concentration of AmB in mice plasma declined

biexponentially and AmB concentrations were signifi-

cantly higher for LN-A and LN-A-MAN than

Fungizone-treated mice (P , 0.05) at all time points.

The pharmacokinetic parameters of LN-A, LN-A-

MAN, and Fungizone were given in Table 2.

Tissue distribution of AmB encapsulated in LN

Tissue distribution after administration of LN-A,

LN-A-MAN, and Fungizone was assessed in the liver,

the spleen, the kidney, and the brain.

Liver

Amphotericin B concentrations in the liver after

i.v. injection of Fungizone, LN-A, and LN-A-MAN

are shown in Figure 1. After 15 min of injection,

AmB concentration in the liver is in the order of

LN-A-MAN . LN-A . Fungizone. There were

large differences in the liver uptake between LN-A,

LN-A-MAN, and Fungizone.

Spleen

Amphotericin B concentrations in the spleen

after i.v. injection of Fungizone, LN-A, and LN-A-

MAN are shown in Figure 2. After 15 min of

injection, AmB concentration in the spleen is in the

order LN-A-MAN . LN-A . Fungizone.

Kidney

Amphotericin B concentrations in the kidney

after i.v. injection of Fungizone, LN-A, and LN-A-

MAN are shown in Figure 3. After 15 min of

injection, the AmB concentration in the kidney is in

the order Fungizone . LN-A . LN-A-MAN.

Brain

Amphotericin B concentration in the brain after

i.v. injection of Fungizone, LN-A, and LN-A-MAN

are shown in the Figure 4. After 15 min of injection,

the AmB concentration in the brain is in the order

LN-A . Fungizone . LN-A-MAN.

Discussion

Fungizone, an intravenously administered colloidal

dispersion of AmB with sodium deoxycholate, use is

limited both by severe and acute toxic side effects, such

as fever, chills, hemolysis, and vomiting and symptoms

of nephrotoxicity, which develop after several weeks of

therapy (Gallis et al. 1990). To overcome these

problems, several strategies have been developed such

as liposomes and lipid-based formulations of AmB.

It is desirable to lower the therapeutic dose of

Fungizone because its elimination from the body is

very slow and repeated administration would lead to

its accumulation (Atkinson and Bennett 1978; Fukui

et al. 2003). Toxicity studies with the formulations of

AmB indicate that these lipid-based formulations are

Table 1. Entrapment efficiency and assay of amphotericin B lipid

nanosphere formulations.

Group Entrapment efficiency (%) Assay (mg/ml)

LN-A 99.64 ^ 0.09 0.91 ^ 0.06

LN-A-MAN 99.50 ^ 0.02 0.93 ^ 0.04

Amphotericin B lipid nanospheres 143

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well tolerated by the animals. The coupling of the

suitable glycosides with amino group of phosphatidyl

ethanolamine were characterized by the liposomes

with regard to the targeting moiety is done by using

glutaraldehyde (Ghosh and Bachhawat 1980). After

i.v. administration, AmB in all three formulations

showed biexponential disposition irrespective of

species. AmB in all three formulations showed similar

pharmacokinetic profile in mice. LN-A showed higher

terminal half-life, which may be due to high protein

binding and slow release of drug from the storage

sites by unknown mechanism (Atkinson and Bennett

1978).

Lipophilic drugs encapsulated in lipid emulsions

showed higher plasma concentration following i.v.

administration than solution forms (Takino et al.

1993). On entering the plasma, the lipid emulsions

rapidly acquire apolipoproteins from circulating

lipoproteins and for such interactions hydrophobic

surface on particles is required (Takino et al. 1994).

AmB in LN-A-MAN was cleared from plasma more

rapidly than other (Table 2). LN-A-MAN is bigger in

Figure 1. Concentration vs. time profiles of amphotericin B in the liver of mice (n ¼ 3).

Figure 2. Concentration vs. time profiles of amphotericin B in the spleen of mice (n ¼ 3).

P. R. VeeraReddy et al.144

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size when compared with LN formulations. It is

reported that reticuloendothelial system (RES)

removes larger particles quickly than smaller particles

(Tuhina et al. 2000).

The liver, the spleen, and the kidney were the

most frequent organs, where fungal infections appear

and the kidney is the organ where AmB produces

toxicity. Higher AmB concentration in the liver

Table 2. Pharmacokinetic parameters following i.v. administration of various formulations of amphotericin B in mice.

Group T1/2a (h) T1/2b (h) AUC0–a (mg h/ml) CL (ml/h/kg) Vss (l kg21) MRT (h) Cmax (mg/ml)

Fungizone 0.42 ^ 0.24 7.7 ^ 2.8 23.7 ^ 8.3 227 ^ 7.7 2.10 ^ 0.34 16.62 ^ 14 6.3 ^ 0.5

LN-A 0.3 ^ 0.01 17.6 ^ 2.8 164.2 ^ 33.9 31 ^ 7.3 0.75 ^ 0.06 24.48 ^ 4.1 20.4 ^ 0.8

LN-A-MAN 0.32 ^ 0.02 21.5 ^ 3.6 229.2 ^ 72.2 23 ^ 6.2 0.67 ^ 0.11 30.2 ^ 5.3 21.6 ^ 0.77

Values in parentheses represent SD (n ¼ 3).

Figure 3. Concentration vs. time profiles of amphotericin B in the kidney of mice (n ¼ 3).

Figure 4. Concentration vs. time profiles of amphotericin B in the brain of mice (n ¼ 3).

Amphotericin B lipid nanospheres 145

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following LN-A-MAN administration indicates that

this formulation may be responsible for higher

antileishmanial activity. However, since the lipid

emulsions injected directly into blood space are

foreign substances to the body, it could not be ruled

out that lipid emulsions are recognized by alternative

pathways of complement activation and captured by

macrophages in RES organs. It has been reported that,

when lipid emulsions are administered by i.v. they are

rapidly taken up by the RES in the liver and the spleen

(Khoo et al. 1994; Daneshmend and Warnock 1983).

Mannose is specifically involved in the recognition of

parasite or appropriate ligands on the macrophage

surface (Goutam et al. 1996). Attempts have been

made to avoid the trapping of lipid nanospheres by the

RES by using surface-modified lipid microspheres

(LM) (Schreoder et al. 1998), small LM (Kreuter et al.

1995) and negatively charged LM (Hultin et al. 1995).

In the case of the kidney, AmB concentration was

higher following Fungizone administration; therefore,

renal toxicity of Fungizone was found to be higher

comparatively than other lipid formulations. All lipid

formulations tested have shown no significant differ-

ence in the kidney concentrations and these results are

supported by the renal toxicity data of these

formulations. The serum creatinine levels of all lipid

formulations were found to be similar. A similar result

had previously been reported with AmB lipid

formulations (Espuelas et al. 1997).

Many colloidal carrier systems have been studied to

achieve blood brain barrier (BBB) penetration of drug

(Gopper and Muller 2003). Higher brain concen-

trations of AmB following LNA administration than

that of Fungizone were observed. AmB in LN-A-MAN

showed lower BBB concentration in comparison with

LN-A. The adsorption pattern of apolipoproteins on

LN-A and LN-A-MAN could be different that

determines the ability to cross BBB (Kreuter et al.

1995). Furthermore, role of smaller size of LN-A

cannot be ruled out.

Conclusion

Based on the results, it is evident that AmB lipid

nanosphere formulations have improved pharmacoki-

netic parameters than Fungizone. LN-A-MAN found

to distribute more rapidly to the liver and the spleen,

which is responsible for observed higher antileishma-

nial activity. AmB concentration following Fungizone

was found to be higher in the kidney, and serum

creatinine levels were also found to be higher

indicating nephrotoxicity. Lipid formulations were

found to be more efficacious and less toxic in

comparison with simple formulation.

Declaration of interest: The authors report no

conflicts of interest.

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