Immune stimulating and therapeutic potential of tuftsin-incorporated nystatin liposomes against Cryptococcus neoformans in leukopenic BALB/C mice

R E S EA RCH AR T I C L E

Immune stimulating and therapeutic potential oftuftsin-incorporated nystatin liposomes against Cryptococcus

neoformans in leukopenic BALB/C mice

Masood Alam Khan1,2, Ahmed Aljarbou2, Arif Khan1,2 & Mohammad Owais1

1Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, India; and 2Department of Pharmaceutics, College of Pharmacy, Qassim

University, Buraidah, Saudi Arabia

Correspondence: Mohammad Owais,

Interdisciplinary Biotechnology Unit,

Aligarh Muslim University, Aligarh-202002,

India. Tel.: + 91 571 2720388;

fax: + 91 571 2721776;

e-mail: [email protected]

Received 28 November 2011; revised 14

March 2012; accepted 13 May 2012.

Final version published online 11 July 2012.

DOI: 10.1111/j.1574-695X.2012.00992.x

Editor: Richard Marconi

Keywords

cryptococcal meningitis; drug delivery;

immunomodulator; nystatin.

Abstract

Cryptococcus neoformans infection is a common fungal infection in persons

infected with human immune deficiency virus (HIV) or those with defective

cell-mediated immunity. Since treatment of cryptococcal meningitis poses a big

challenge, the present study aimed to develop a novel liposomal therapeutic

formulation against cryptococcosis. Treatment with tuftsin-incorporated lipo-

somes increased the anti-cryptococcal activity of murine peritoneal macro-

phages. Prophylactic treatment of mice with tuftsin-incorporated liposomes

reduced the dissemination of C. neoformans to brain tissues. Moreover, the

co-administration of tuftsin with nystatin liposomes augmented the anti-

cryptococcal activity of nystatin, as mice treated with tuftsin-incorporated

nystatin liposomes showed the highest survival and least fungal burden in

their brain tissues. The results of the present study favour the use of immune-

stimulating molecules along with antifungal agents in the treatment of oppor-

tunistic fungal infections.

Introduction

The opportunistic fungal pathogens such as Candida,

Aspergillus, Cryptococcus and Histoplasma spp. occur most

commonly in patients with impaired body immunity

(Ruhnke, 2004). The emergence of HIV infections, the

use of anti-cancer chemotherapy and intra-vascular

devices, and the persistent use of antibiotics and immu-

nosuppressive drugs for organ transplantation have

increased the frequency of fungal infections (Vilchez

et al., 2002; Kontoyiannis et al., 2003; Olszewski et al.,

2010). Most fungal pathogens cause diseases in immuno-

compromised subjects, but Cryptococcus neoformans can

cause infection in both immunocompetent and immuno-

compromised individuals (Rude et al., 2002). Cryptococcus

neoformans adopts unique virulence factors and strategies

to evade the host immune mechanisms (Olszewski et al.,

2010; Voelz & May, 2010).

Cryptococcus neoformans causes mild to severe pneu-

monia-like disease depending upon the virulence of the

isolate as well as on the status of the host immune sys-

tem (Mitchell & Perfect, 1995). Host resistance to

C. neoformans is largely dependent on the active partici-

pation of the innate and adaptive immune responses

(Shoham & Levitz, 2005; Zhou & Murphy, 2006). Anti-

bodies are less effective in elimination of C. neoformans,

but they function as opsonins in antibody-dependent

cellular cytotoxicity (Casadevall & Pirofski, 2005). The

cell-mediated immune response has been shown to play

a crucial role in controlling Cryptococcus infection (Sho-

ham & Levitz, 2005; Olszewski et al., 2010; Voelz &

May, 2010).

Liposomal formulations of polyene antibiotics such as

amphotericin B and nystatin have been proved to be the

most effective agents in the treatment of various fungal

infections (Nq et al., 2003). However, the toxicities

caused by polyene antibiotics limit their use in a clinical

setting. Liposomal formulations minimize the toxic mani-

festations associated with these drugs and are widely used

in the treatment of deep-seated fungal infections

(Dupont, 2002; Nasti et al., 2006). Moreover, liposomal

nystatin was an effective treatment against aspergillosis in

ª 2012 Federation of European Microbiological Societies FEMS Immunol Med Microbiol 66 (2012) 88–97Published by Blackwell Publishing Ltd. All rights reserved

IMM

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patients not responding to or intolerant of amphotericin

B therapy (Offner et al., 2004).

Tuftsin is a tetrapeptide (Thr-Lys-Pro-Arg) fraction of

the immunoglobulin G (IgG) molecule (Siemion &

Kluczyk, 1999). It binds to neutrophils and macrophages

to stimulate their phagocytic activity (Siemion & Kluc-

zyk, 1999) and also increases tumor necrosis factor

alpha (TNF-a) release from human Kupffer cells (Siem-

ion & Kluczyk, 1999). Tuftsin-macrophage interaction

results in activation of macrophages expressing nitric

oxide (NO) synthase to produce NO. Murine peritoneal

macrophages activated by tuftsin were able to kill the

intracellular protozoan Leishmania major (Siemion &

Kluczyk, 1999).

The use of immunomodulator-based therapy could be

of great significance in the successful treatment of infec-

tious diseases caused by opportunistic fungal pathogens

(Casadevall & Pirofski, 2001; Romani, 2001; Hamad,

2008). The immune-stimulatory agents not only help to

restore the normal immune response in immunocompro-

mised hosts, but also increase the efficacy of anti-

microbial agents (Kullberg et al., 2004; Matthews &

Burnie, 2004). Because of the presence of its receptors on

macrophages, liposomized-tuftsin facilitates the targeting

of drugs to macrophages, which helps eliminate the intra-

cellular infections (Gupta & Haq, 2005). The use of tuft-

sin has been shown to increase the efficacy of antibiotics

against protozoan, bacterial and fungal infections (Agar-

wal et al., 1994; Agrawal et al., 2002; Khan et al., 2003).

Tuftsin-incorporated nystatin liposomes have been shown

to be more effective, safer and more stable than conven-

tional nystatin liposomes without tuftsin (Khan et al.,

2006). In the present study, we showed that liposomal

tuftsin reduces the dissemination of C. neoformans in the

brain tissues of leukopenic mice.

Materials and methods

Mice

BALB/C mice used in this study were purchased from

National JALMA Research Institute for Leprosy, Agra,

India. The techniques used for bleeding, injection and

sacrifice of animals were approved by the Animal Ethics

Committee [Committee for the Purpose of Control and

Supervision of Experiments on Animals (CPCSEA), Gov-

ernment of India].

Materials

Nystatin was purchased from Sigma Chemical Co.

(St. Louis, MO). Cholesterol was bought from Centron

Research Laboratory (Bombay, India) and used after

crystallization with methanol. Egg phosphatidylcholine

(egg PC) was isolated and purified as described earlier

(Singleton et al., 1965). Tuftsin modified at the C-termi-

nus was prepared as described earlier (Gupta & Haq,

2005).

Test strain

The clinical isolate of C. neoformans was obtained from

the Department of Microbiology, J. N. Medical College,

A.M.U. Aligarh, India, as described earlier (Nasti et al.,

2006). The isolate was subcultured on Sabouraud Dex-

trose Agar at 37 °C for 48–72 h.

Antifungal susceptibility testing

The minimum inhibitory concentration (MIC) of nystatin

was determined by the broth macrodilution method as

described by the National Committee for Clinical Labora-

tory Standards recommendations (NCCLS, 1997). Nystatin

was tested over a concentration range of 0.02–5 lg mL�1

as described earlier (Nasti et al., 2006) Testing was

performed in 96-well round-bottom microtitre plates. A

cell suspension of C. neoformans was prepared in RPMI-

1640 medium and adjusted to give a final inoculum of

2 9 103 cells mL�1. The wells containing fungal inoculum

with different concentrations of the nystatin and proper

controls were incubated for 48 h.

Quantitative analysis of leukocytes

The number of leukocytes was determined by total leuko-

cyte count (TLC). The cells were counted on day 3 after

cyclophosphamide treatment in both control and tuftsin-

treated mice. Blood samples (20 lL) were taken from

mice and mixed with Turk’s fluid (380 lL) and the total

number of leukocytes was counted using a Neubaur

chamber (Brown, 1980).

Preparation of tuftsin-incorporated and

tuftsin-free nystatin liposomes

Liposomes were prepared from egg PC (49 lmol) and

cholesterol (21 lmol) with or without modified tuftsin

(7–8% by PC weight) by the sonication method (Owais

& Gupta, 2000). All the ingredients including nystatin

(Drug: Lipid, 1 : 20 molar ratio) were dissolved in a

round-bottomed flask in a mixture of chloroform/metha-

nol (1: 1, v/v). The solvents were evaporated under

reduced pressure to form a thin film of lipids and the

traces of the solvents were removed by subjecting the

flask to vacuum. Subsequently, the lipid film (consisting

of egg PC/cholesterol, nystatin) was hydrated with 2.0 mL

FEMS Immunol Med Microbiol 66 (2012) 88–97 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Tuftsin liposomes control murine cryptococcosis 89

of sterile normal saline with intermittent stirring followed

by sonication (1 h, 4 °C) in a bath type sonicator under

N2 atmosphere. After sonication, the lipid formulations

of drug were centrifuged at 10 000 g for 1 h at 4 °C to

remove traces of undispersed lipids, and finally dialyzed

against normal saline for 24 h at 4 °C in the dark.

Determination of size, lamellerity and

structure of liposomes

The liposome lamellerity, size and structure were evalu-

ated by transmission electron microscopy as described

earlier (Khan et al., 2006).

Estimation of liposome-incorporated nystatin

and tuftsin

Nystatin incorporation in liposomes was determined

spectrophotometrically and by high performance liquid

chromatography (HPLC) as described earlier (Khan et al.,

2006). A standard curve of nystatin was plotted at

320 nm. Nystatin associated with liposomes was esti-

mated by dissolving the liposomal formulation in metha-

nol and determining the absorbance at 320 nm using an

equal amount of lipid in methanol as a blank. The

amount of nystatin entrapped in liposomes was calculated

from the standard curve of the drug. The intercalation

efficiency of nystatin in liposomes was also estimated by

the HPLC method. Briefly, the sample (20 lL) was

injected in a Hypersil octyldecyl-silane 5 lm particle size

analytical column (150 9 4.6 mm internal diameter).

A standard curve was plotted by calibrating peak area

vs. amount of the drug injected in the column. The

intercalation efficiency of nystatin in both plain egg PC

and tuftsin-bearing liposomes was found out to be of the

same order as estimated by spectrophotometrically

(90 ± 4%).

To facilitate the incorporation of tuftsin into liposome

bilayers, tuftsin was modified at its C-terminus by attach-

ing a long fatty acyl residue through an ethylenediamine

spacer arm [Thr-Lys-Pro-Arg-NH-(CH2)2-NH-COC15H31]

according to the published procedure (Singhal et al.,

1984). The tuftsin incorporated in nystatin liposomes was

estimated by the bicinchoninic acid (BCA) method.

Briefly, the liposomes (given volume) were lysed with 10%

Triton X-100 solution (the final concentration of Triton

X-100 was maintained at 1%). The mixture of solutions A

(containing sodium carbonate, sodium bicarbonate,

BCA and sodium tartrate in 0.1 M sodium hydroxide)

and B (4% cupric sulphate) of BCA reagent was added

to an aliquot of tuftsin-incorporated liposomes digested

with Triton X-100. The reaction mixture was incubated

at 37 °C for 45 min. The absorbance was measured at

600 nm and the content of tuftsin incorporated in lipo-

somes was determined using a standard curve of tuftsin

plotted in the presence of Triton X-100. The percent

intercalation efficiency of the incorporated tuftsin was

found to be about 95%.

Preparation of C. neoformans cells for

infection

Yeast cells of C. neoformans were harvested from agar

plates into YPD (1% yeast extract, 2% peptone, 5% dex-

trose) medium at 37 °C for 48 h as described earlier (Nasti

et al., 2006). The cells were washed with sterile normal

saline at low speed centrifugation (670 g) and diluted to

the appropriate concentrations in saline prior to use. Each

mouse was infected intravenously with 1 9 105 cells of

C. neoformans to determine the kinetics of cryptococcal

meningitis in mice. In survival studies, each mouse was

infected with 7 9 105 cells of C. neoformans.

Assessment of anticryptococcal activity of

liposomal tuftsin-treated macrophages

Macrophages (1 9 105 cells per well) were seeded in trip-

licates in 24-well costar plates containing complete med-

ium supplemented with 10% fetal bovine serum as

described earlier (Khan et al., 2003). The plates were

incubated at 37 °C in 5% CO2 for 24 h. Medium was

taken out and cells were washed. The macrophages were

then treated with sham liposomes or tuftsin-bearing lipo-

somes (the concentration of tuftsin at 1 lg mL�1) for

4 h. Cells were washed and C. neoformans (5 9 104

cells per well) was added to tuftsin-treated or untreated

macrophages and incubated for 2 h. After 2 h of infec-

tion, non-phagocytosed C. neoformans cells were rinsed

with cold phosphate-buffered saline (PBS). The number

of colony forming units (CFUs) recovered from the lysis

of macrophages (no tuftsin treatment) after 2 h of infec-

tion was considered baseline. After 24 h of incubation,

macrophages were lysed using 0.1% Tween-20 to deter-

mine the numbers of phagocytosed C. neoformans cells.

The various dilutions of lysates were spread on SD agar

plates and incubated at 37 °C for 48 h.

Induction of leukopenia in mice

Mice were injected intravenously with a single dose of

cyclophosphamide (250 mg kg�1) to induce leukopenia.

Leukopenia temporarily persisted for a week and mice

were infected on day 3 post-cyclophosphamide injection.

Day 0 was the day of infection of mice with C. neofor-

mans. In prophylactic studies, pretreatment with tuftsin

(50 lg per mouse, intraperitoneal) was started 8 h after


90 M.A. Khan et al.

cyclophosphamide treatment for three consecutive days

(day �3 to day �1).

Kinetics of the dissemination of C. neoformans

in the brain of immunocompetent and

leukopenic mice pretreated with sham

liposomes or tuftsin-incorporated liposomes

The prophylactic role of immunomodulator tuftsin

against C. neoformans was investigated in immunocompe-

tent and leukopenic mice. Mice were treated with lipos-

omized-tuftsin (50 lg per mouse) intraperitoneally. Each

mouse was infected with C. neoformans (1 9 105 cells per

mouse in a volume of 100 lL sterile saline) by the intra-

venous route on day 3 post-cyclophosphamide treatment.

Brain tissues were collected aseptically from infected mice

at 3, 6, 12, 24 and 48 h post-C. neoformans infection. The

organs were washed with sterile PBS and homogenized in

sterile PBS. Appropriate dilutions of homogenates were

plated (100 lL) in triplicate on chloramphenicol contain-

ing Sabouraud-dextrose agar plates and CFU were

counted after 48 h of growth at 37 °C. Chloramphenicol

was used to prevent any bacterial contamination.

Treatment with tuftsin-incorporated or

tuftsin-free nystatin liposomes

Immunocompetent and leukopenic mice were administered

tuftsin-incorporated or tuftsin-free nystatin liposomes

intraperitoneally on days 1, 3 and 5 post-Cryptococcus

infection. Each treatment group contained 10 mice, divided

into the following groups: (1) saline, (2) sham liposomes, (3)

tuftsin liposomes, (4) liposomal nystatin (3 and 5 mg kg�1)

and (5) tuftsin-incorporated liposomal nystatin (3 and

5 mg kg�1).

Prophylactic use of liposomal tuftsin followed

by treatment with tuftsin-free or tuftsin-

incorporated nystatin liposomes against

C. neoformans in leukopenic mice

Since treatment with tuftsin-free or tuftsin-incorporated

nystatin liposomes did not completely cure C. neoformans-

infected leukopenic mice, the prophylactic effect on the

efficacy of liposomal nystatin in leukopenic mice of tuft-

sin was determined. Each mouse was treated with 50 lgof liposomized-tuftsin intraperitoneally for three con-

secutive days as described earlier (Khan et al., 2003).

Pretreatment with tuftsin liposomes (PT-tuftsin-liposomes)

in leukopenic mice was started 8 h after cyclophospha-

mide injection. After 3 days of tuftsin treatment, each

mouse was infected with 5 9 105 cells of C. neoformans.

Mice were divided into the following groups: (1) saline,

(2) sham liposomes, (2) PT-tuftsin-liposomes + sham

liposomes, (4) tuftsin liposomes, (5) PT-tuftsin-liposomes +tuftsin liposomes, (6) liposomal nystatin (5 mg kg�1), (7)

PT-tuftsin-liposomes + liposomal nystatin (5 mg kg�1),

(8) tuftsin-bearing liposomal nystatin (5 mg kg�1), (9) PT-

tuftsin-liposomes + tuftsin-liposomal nystatin (5 mg kg�1).

Quantitative analysis of C. neoformans in the

brain tissues

The prophylactic and therapeutic role of tuftsin was evalu-

ated by determining the fungal load in brain tissues. Three

mice from each group were sacrificed on day 3 post-

C. neoformans infection (8 h after the 2nd dose of treat-

ment) and their brains were taken out aseptically as

described earlier (Nasti et al., 2006). Briefly, weighed

portions of the brain tissues were homogenized in 5 mL of

sterile normal saline and different dilutions of the suspen-

sion were plated on SD agar plates containing chloramphe-

nicol. The plates were incubated at 37 °C for 48–72 h. The

numbers of colonies were counted and the fungal load was

determined by multiplying them by the dilution factor.

Statistical analyses

Analysis of the survival of mice was performed using

Kaplan–Meier curve, and various groups were compared

by log-rank test. Fungal burden in organs was analyzed

by one-way ANOVA followed by the Bonferroni post hoc

test using GRAPHPAD PRISM software version 3.0.

Results

In vitro antifungal susceptibility testing

The minimum inhibitory concentration (MIC) was defined

as the lowest concentration of nystatin at which there was

complete inhibition of the fungal growth. The MIC of

nystatin for C. neoformans was found to be 1.5 lg mL�1.

Tuftsin facilitates the early recovery of the

depleted leukocytes in cyclophosphamide-

treated mice

Immuno-potential effect of tuftsin was analyzed by treating

leukopenic mice with tuftsin-bearing liposomes, and mice

treated with saline or sham liposomes acted as controls.

The number of leukocytes was counted in the blood of

tuftsin-treated or untreated mice. Our results demonstrated

that treatment with tuftsin liposomes induced early recov-

ery of leukocytes in leukopenic mice. We found that leuko-

penic mice treated with tuftsin-liposomes have higher

counts of leukocytes (1220–1956) on day 4 post-cyclophos-



phamide injection compared with mice treated with saline

(166–272) or sham liposomes (183–344) (P < 0.001).

Moreover, the group of leukopenic mice treated with

tuftsin-incorporated liposomes showed complete recovery

of leukocytes on day 8 post-cyclophosphamide treatment,

whereas saline- or sham liposome-treated mice showed

only 40–60% recovery in leukocyte counts.

Tuftsin-treated macrophages show more

resistance against C. neoformans

Cryptococcus neoformans can multiply inside macrophages

as shown by an increase in the numbers of CFUs after

24 h of infection (Fig. 1). Tuftsin binds to macrophages

and activates them to kill the phagocytosed C. neoformans.

Liposomized-tuftsin showed an increased effect on macro-

phage-mediated killing of intracellular pathogens, as tuft-

sin-incorporated liposomes were taken up rapidly by

macrophages compared with similar liposomes without

tuftsin. Macrophages treated with tuftsin-incorporated

liposomes showed increased anti-cryptococcal activity

compared with tuftsin-free liposomes.

Treatment with tuftsin-liposomes reduces the

dissemination of C. neoformans to brain

tissues of immunocompetent and

leukopenic mice

The kinetics of C. neoformans infection to the brain

tissues was determined in immunocompetent and

leukopenic mice prophylactically treated or untreated

with tuftsin-incorporated liposomes. Mice were eutha-

nized and brain tissues were taken at 3, 6, 12, 24 and

48 h post-C. neoformans infection. There was a reduced

C. neoformans burden in the brain tissues of mice treated

with tuftsin-incorporated liposomes compared with mice

treated with sham liposomes or saline (Fig. 2a).

The C. neoformans infection was found to be more

severe in leukopenic mice compared with immunocom-

petent mice as shown by higher numbers of CFUs in the

brain tissues of leukopenic mice (Fig. 2b). Leukopenic

mice pretreated with tuftsin-incorporated liposomes also

showed reduced burden of C. neoformans in the brain

tissues, particularly at 24 and 48 h post-infection

(Fig. 2b). This suggests that activation of the immune

system by tuftsin liposomes plays an important role in

reducing the dissemination of C. neoformans to the brain

tissues.

Incorporation of tuftsin augments the activity

of liposomal nystatin against C. neoformans in

BALB/C mice

The tuftsin-incorporated liposomal nystatin at the dose

of 3 and 5 mg kg�1 on days 1, 3 and 5 post-infection

showed increased efficacy against systemic C. neoformans

in both immunocompetent and leukopenic mice. Immu-

nocompetent mice treated with tuftsin-incorporated nys-

tatin liposomes (5 mg kg�1) showed 100% survival rate

on day 40 post-infection, whereas the mice treated with

liposomized-nystatin (5 mg kg�1) without tuftsin showed

a 60% survival rate (P = 0.029; Fig. 3a). Mice treated with

tuftsin-incorporated liposomal nystatin at the dose of

3 mg kg�1 showed a 70% survival, whereas liposomized-

nystatin at the same dose showed only 30% survival.

Thus tuftsin-incorporated nystatin liposomes showed

superiority over tuftsin-free nystatin liposomes in treating

C. neoformans-infected mice.

Cryptococcus neoformans-infected leukopenic mice were

also treated with tuftsin-free or tuftsin-incorporated liposo-

mal nystatin on days 1, 3 and 5 post-infection. Like immu-

nocompetent mice, leukopenic mice showed an increased

survival upon treatment with tuftsin-incorporated nystatin

liposomes. The group of leukopenic mice treated with tuft-

sin-incorporated nystatin liposomes (5 mg kg�1) showed a

60% survival rate on day 40 post-C. neoformans challenge,

whereas mice treated with tuftsin-free nystatin liposomes at

the same dose showed only 20% survival (Fig. 3b). Mice

treated with tuftsin-incorporated nystatin liposomes at

3 mg kg�1 showed a 30% survival rate, whereas mice trea-

ted with tuftsin-free nystatin liposomes (3 mg kg�1) died

before day 40 post-infection (Fig. 3b). These results showed

that incorporation of tuftsin on the surface of nystatin

Fig. 1. Treatment of macrophages with tuftsin liposomes increases

the anti-cryptococcal activity of macrophages. Peritoneal macrophages

were untreated or treated with sham liposomes or tuftsin liposomes

for 2 h as described in Materials and methods. Macrophages were

infected with Cryptococcus neoformans for 24 h. Cells were lysed

and intracellular yeasts were plated on Sabouraud dextrose agar for

48 h at 37 °C. Medium vs. tuftsin liposomes (P < 0.05); Sham

liposomes vs. tuftsin liposomes (P < 0.05).


92 M.A. Khan et al.

liposomes also increased their efficacy compared with con-

ventional nystatin liposomes in leukopenic mice.

The severity of C. neoformans infection was assessed by

culturing the brain tissue homogenates of the infected

mice. Mice treated with tuftsin-incorporated liposomes

(no nystatin) showed reduced fungal burden in brain tis-

sues compared with mice treated with sham liposomes

(Fig. 4a; P < 0.001). As evident from Fig. 2, the incorpo-

ration of tuftsin in nystatin liposomes showed increased

anti-cryptococcal activity compared with tuftsin-free nys-

tatin liposomes. Tuftsin-incorporated nystatin liposomes

(3 mg kg�1) showed superior efficacy compared with

similar liposomal formulation of nystatin without tuftsin

(P < 0.05) in immunocompetent mice (Fig. 4a).

Although the fungal burden was found to be very high

in the brain tissues of leukopenic mice, tuftsin-incorporated

liposomes were more effective in reducing C. neoformans

burden from brain tissues of leukopenic mice (P < 0.05;

Fig. 4b). Tuftsin-incorporated liposomal nystatin at the

dose of 3 mg kg�1 was found to possess superior efficacy

against C. neoformans than same formulation without

tuftsin (P < 0.01; Fig. 4b).

Prophylactic use of tuftsin-liposomes increases

the efficacy of tuftsin-free or tuftsin-

incorporated nystatin liposomes in leukopenic

mice

As leukopenic mice showed more severe C. neoformans

infection and most of the mice died before day 40 post-

infection, it might be important to analyze the effect of

tuftsin pretreatment on these mice before challenging

them with infection. Pretreatment with tuftsin liposomes

followed by post-infection treatment with tuftsin-incorporated

liposomal nystatin (5 mg kg�1) showed a 90% survival

rate (Fig. 5a). Leukopenic mice without any pretreatment

followed by treatment with the same dose of tuftsin-free

liposomal nystatin showed only 20% survival (P = 0.003).

Fig. 2. Liposomal tuftsin reduces the dissemination of Cryptococcus neoformans to the brain tissues of BALB/C mice. (a) Immunocompetent and

(b) Leukopenic BALB/C mice were intravenously infected with 1 9 105 cells of C. neoformans and brains were taken out aseptically on 3, 6, 12,

24 and 48 h post-infection. The fungal burden was calculated by culturing brain tissue homogenates. Results are expressed as mean data from

three mice in each group ± SD. *P < 0.05 as compared with sham liposomes and saline at the respective times.

Fig. 3. Treatment with tuftsin-incorporated nystatin liposomes increases the survival of Cryptococcus neoformans-infected immunocompetent

and leukopenic BALB/C mice. (a) Immunocompetent and (b) leukopenic mice were intravenously infected with 7 9 105 cells of C. neoformans

(day 0). Treatment with tuftsin-incorporated or tuftsin-free liposomized-nystatin (3 or 5 mg kg�1) was given at days 1, 3 and 5 post-infection.

Saline (♦), sham liposomes (♢), tuftsin liposomes (▲), liposomal nystatin (3 mg kg�1) (●), liposomal nystatin (5 mg kg�1) (■), tuftsin-

incorporated liposomal nystatin (3 mg kg�1) (○), tuftsin-incorporated liposomal nystatin (5 mg kg�1) (□).



Pretreatment with liposomal tuftsin also increases the sur-

vival of C. neoformans infected leukopenic mice from

20% to 60% after treatment with liposomal nystatin

(5 mg kg�1) (Fig. 5a).

The effect of tuftsin pretreatment on the therapeutic

efficacy of tuftsin-free or tuftsin-incorporated liposomal

nystatin was also assessed by culturing the brain tissue

homogenates. The fungal burden was found to be the

least in mice pretreated with tuftsin liposomes followed

by treatment with tuftsin-incorporated liposomal nystatin

(Fig. 5b). Tuftsin pretreated leukopenic mice showed

greater resistance to C. neoformans infection compared

with mice not pretreated with tuftsin (P < 0.01). The

fungal burden was much reduced in the brain tissues of

Fig. 4. Treatment with tuftsin-incorporated nystatin liposomes showed higher efficacy in reducing fungal burden from the brain tissues of

Cryptococcus neoformans-infected immunocompetent and leukopenic BALB/C mice. (a) Immunocompetent and (b) leukopenic mice were

infected with 7 9 105 cells of C. neoformans (day 0). Treatment with tuftsin-incorporated or tuftsin-free liposomized-nystatin (3 or 5 mg kg�1)

was given at days 1, 3 and 5 post-infection. On day 3, three mice from each group were sacrificed and their brains were taken out and

homogenized. The brain tissue homogenates were cultured to determine fungal load. For immunocompetent mice: sham liposomes vs. tuftsin

liposomes (P < 0.001); liposomal nystatin (3 mg kg�1) vs. tuftsin-incorporated liposomal nystatin (3 mg kg�1) (P < 0.05). For leukopenic mice:

sham liposomes vs tuftsin liposomes (P < 0.05); liposomal nystatin (3 mg kg�1) vs. tuftsin-incorporated liposomal nystatin (3 mg kg�1) (P < 0.01).

Fig. 5. Prophylactic use of tuftsin-incorporated liposomes increases the efficacy of liposomal nystatin against Cryptococcus neoformans in

leukopenic BALB/C mice. (a) Leukopenic mice were pretreated with liposomized-tuftsin (50 lg per mouse) by the intraperitoneal route for three

consecutive days. Mice were infected with 7 9 105 cells of C. neoformans (day 0). Treatment with tuftsin-incorporated or tuftsin-free

liposomized-nystatin (5 mg kg�1) was given at days 1, 3 and 5 post-infection. Mice were observed for 40 days to check their survival. Saline (9),

sham liposomes (□), PT-tuftsin + sham liposomes (■), tuftsin liposomes (D), PT-tuftsin + tuftsin liposomes (▲), liposomal nystatin (5 mg kg�1)

(∇), PT-tuftsin + liposomal nystatin (5 mg kg�1) (▼), tuftsin-incorporated liposomal nystatin (5 mg kg�1) (♢), PT-tuftsin + tuftsin-incorporated

liposomal nystatin (5 mg kg�1) (♦). (b) On day 3, three mice from each group were sacrificed and their brains were taken out and homogenized.

The brain tissue homogenates were cultured to determine fungal load. Liposomal nystatin vs. PT-tuftsin + liposomal nystatin (P < 0.01),

Liposomal nystatin vs. PT-tuftsin + tuftsin-incorporated liposomal nystatin (P < 0.001).


94 M.A. Khan et al.

leukopenic mice pretreated with tuftsin liposomes fol-

lowed by therapy with tuftsin-incorporated liposomal

nystatin compared with mice treated with liposomal nys-

tatin without tuftsin (P < 0.01).

Residual fungal burden

To determine the clearance of infection, the residual

fungal burden in the brain tissues of the surviving mice

was analyzed on day 40 post-C. neoformans infection. It

fell in the range of 0–75 CFUs per brain, which suggested

that infection in these mice had subsided.

Discussion

Among the fungal pathogens, C. neoformans is one of the

leading causes of morbidity and mortality in immunologi-

cally compromised persons (Zhou & Murphy, 2006). The

severity of the infection and disease depends largely on the

competence of the host immune system, inoculum and the

virulence of the pathogen. Neutrophils and macrophages

play an important role in controlling C. neoformans infec-

tion (Romani, 2001). On the other hand, in order to estab-

lish itself, C. neoformans tries to modulate or subvert the

host immune system to establish the infection (Vecchiarelli,

2007).

Tuftsin is known to stimulate the phagocytic activity of

neutrophils and macrophages, which play a role in the

protection against fungal infections (Siemion & Kluczyk,

1999). Tuftsin was incorporated on the surface of lipo-

somes. The results of the present study show that treat-

ment with tuftsin-incorporated liposomes reduces the

dissemination of C. neoformans to brain tissues. There

was a reduced burden of C. neoformans in the brain

tissues of mice prophylactically treated with tuftsin lipo-

somes compared with mice treated with liposomes with-

out tuftsin (Fig. 1a). Similar results were found in

cyclophosphamide-treated mice, although the severity of

C. neoformans infection was greater in them (Fig. 1b). As

tuftsin activates the phagocytic activities of neutrophils

and macrophages (Siemion & Kluczyk, 1999), it is quite

possible that neutrophils and macrophages control the

dissemination of C. neoformans to brain tissues of tuftsin-

pretreated mice.

The use of immunomodulators in combination with

chemotherapy imparts greater protection against fungal

infections (Dutta, 2002; Khan et al., 2003). Immunomod-

ulator tuftsin stimulates the respiratory burst phenome-

non in macrophages and neutrophils by producing free

nitrogen and oxygen species (Siemion & Kluczyk, 1999).

Tuftsin-incorporated liposomal nystatin at a dose of

5 mg kg�1 was found to produce a 100% cure rate

against systemic lethal infection of C. neoformans in

immunocompetent mice, whereas C. neoformans-infected

mice treated with liposomal nystatin (without tuftsin) at

the same dose showed only 60% survival. The fungal bur-

den also supported the survival data, as C. neoformans-

infected mice treated with tuftsin-incorporated nystatin

liposomes showed the fewer CFUs in brain tissues.

As opportunistic fungal infections are commonly found

in immunocompromised persons, mice were made leuko-

penic by injecting cyclophosphamide. Quantitative or qual-

itative defects in neutrophils are predisposing factors to

disseminated fungal infections. Neutrophils play a very

important role in limiting the early multiplication of the

fungal pathogens, and during the infection macrophages in

the tissues become activated and prevent the dissemination

of the infection. Leukopenic mice treated with tuftsin-

incorporated nystatin liposomes at the doses of 3 and

5 mg kg�1 showed a 30 and 60% survival rate, respectively,

on day 40 post-C. neoformans infection, whereas the group

of mice treated with nystatin liposomes (without tuftsin) at

the dose of 5 mg kg�1 showed only 20% survival. The

increased severity of C. neoformans infection may be due to

reduced numbers of leukocytes in cyclophosphamide-

injected mice. The results of the present study showed that

combination therapy of tuftsin with liposomal nystatin is

more effective in increasing the survival of C. neoformans-

infected leukopenic mice. The increased effect of tuftsin-

incorporated liposomal nystatin against C. neoformans in

leukopenic mice can be ascribed to the tuftsin-induced

activation of tissue macrophages and residual leukocytes.

Moreover, tuftsin-incorporated nystatin liposomes can

specifically target the drug to macrophages, which may act

as drug depots and reach the site of infection. Thus the

status of innate immune cells at the time of infection plays

a role in determining the anti-cryptococcal efficacy of lipo-

somal nystatin in tuftsin-treated or untreated leukopenic

mice. Tuftsin-incorporated nystatin liposomes stay in the

systemic circulation for a prolonged time, in comparison

with the tuftsin-free nystatin liposomes (Khan et al., 2006).

The present study shows the increased efficacy of liposomal

nystatin in leukopenic mice pretreated with tuftsin-incor-

porated liposomes. This may be due to the stimulating

effect of tuftsin in the recovery of leukocytes or activated

state of tissue macrophages or residual neutrophils in

leukopenic mice. Tuftsin-macrophage interaction results in

the activation of macrophages expressing NO synthase to

produce NO, which plays an important role in killing intra-

cellular C. neoformans (Ghosn et al., 2006). Inducible nitric

oxide synthase-deficient mice showed more susceptibility

to C. neoformans infection compared with control mice

(Aguirre & Gibson, 2000).

The results of the present study clearly demonstrate the

important role of tuftsin in augmenting the anti-crypto-

coccal activity of liposomal nystatin in murine model



because of its ability to specifically target nystatin to intra-

cellular residing sites of the pathogen in macrophages.

Tuftsin also has the ability simultaneously to activate neu-

trophils, monocytes and macrophages for increased phago-

cytosis and killing of pathogens by these cells. Furthermore,

tuftsin can also be used to rejuvenate the immune system

of the patients suffering from immune-deficiency diseases

or undergoing cancer chemotherapy.

Acknowledgements

We are very grateful to Aligarh Muslim University and

Interdisciplinary Biotechnology Unit (IBU) for providing

all the necessary facilities. We are also grateful to Prof.

Abida Malik, Department of Microbiology, JNMC,

Aligarh, for identification and characterization of the

Cryptococcus neoformans isolate. Masood A. Khan thanks

the University Grant Commission (UGC), Govt. of India

for awarding financial assistance in the form of research

fellowship. There is no conflict of interest among authors.

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Immune stimulating and therapeutic potential of tuftsin-incorporated nystatin liposomes against Cryptococcus neoformans in leukopenic BALB/C mice