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Antimicrobial Agents and Chemotherapy, February 1998, p. 348-351, Vol. 42, No. 2
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A Novel Peptide-Grafted Liposomal Delivery System
Targeted to Macrophages
Goutam
Banerjee,
Swapna
Medda, and
Mukul K.
Basu*
Biomembrane Division, Indian Institute of
Chemical Biology, Calcutta 700 032, India
Received 21 October 1996/Returned for modification 28 January
1997/Accepted 3 July 1997
 |
ABSTRACT |
The interaction of chemotactic peptide (e.g., fMet-Leu-Phe)-grafted
liposomes with macrophages is noted to be rapid and specific. At a grafted peptide concentration of 100 nmol, internalization of the
peptide-grafted liposomes by the macrophages is found to reach
equilibrium in 30 min. The peptide alone and the peptide-grafted empty
liposomes are found to show moderate antileishmanial activity in vitro.
Primaquine, which is known to generate O2
in
phagocytic cells, showed leishmanicidal properties when it was tested
in vitro against parasite-infected macrophages over a certain
range of concentrations. It showed much better efficacy against
experimental leishmaniasis when it was used in the fMet-Leu-Phe-grafted liposomal form in comparison with its efficacy when it was either in
the free form or encapsulated in ungrafted liposomes. The conventional toxicity parameters (e.g., blood pathology and tissue
histology-specific enzyme levels related to normal liver function) are
found to be very close to normal when fMet-Leu-Phe-grafted liposomal
primaquine is used. The biodegradabilities of both the drug and the
delivery systems are also found to be very satisfactory. Thus, this
delivery system may have possible applications for the treatment of
leishmaniasis as well as other macrophage-associated disorders.
| |
INTRODUCTION |
Macrophages are chemotactically
responsive cells that are of central importance to both the recognition
and the effector limbs of the immune response. Chemotaxis, by
definition, is the directed movement of cells along a chemical
gradient, and it appears to be an important mechanism by which
inflammatory cells accumulate at local sites (18, 19). The
chemotactic agonists stimulate the macrophages and induce the
respiratory burst (2, 3), resulting in the activation of
NADPH oxidase, which catalyzes the conversion of molecular oxygen to
superoxide anion (O2). This O2 is
the precursor of a series of microbicidal products (6). This
activation is initiated by the binding of a chemotactic agonist to its
receptor. It is prevented by the antagonist and is interrupted by
agonist displacement, indicating that the agonist receptor complex must
persist (3). We have developed a delivery system using one
such chemotactic agonist, i.e., one peptide
(N-formyl-methionine-leucine-phenylalanine [fMLP]) that
has the chemoattracting properties for macrophages. Here we
report the feasibility of applying fMLP-grafted liposomes as a delivery
system for primaquine against experimental leishmaniasis in animal
models.
| |
MATERIALS AND METHODS |
Materials.
Phosphatidylethanolamine (PE), cholesterol,
phosphatidic acid, fMLP, and 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDC1) were purchased from
Sigma. RPMI 1640 medium and medium 199 were from Gibco. All other
reagents were of analytical grade.
Peritoneal macrophages were isolated from Swiss albino mice 4 days after the intraperitoneal injection of 1 ml of sodium
thioglycolate (4%). They were cultured in RPMI 1640 medium containing
L-glutamine (4 mM), HEPES (25 mM), streptomycin (100 µg/ml), penicillin (100 U/ml), and 20% fetal bovine serum. The
amastigotes of Leishmania donovani AG83 were maintained in
susceptible golden hamsters. The promastigotes were obtained after 5 days of continuous culture of the amastigotes in medium 199 containing
20% fetal calf serum, HEPES (0.15 M), penicillin (100 U/ml), and
streptomycin (100 µg/ml). The bovine serum albumin (BSA) was
radioiodinated with chloramine T by the standard method (10)
and was purified from unreacted Na125I by Sephadex G-50
column chromatography.
Preparation of liposomes.
The liposomes were prepared by
following the standard procedure (8). In brief, PE,
cholesterol, and phosphatidic acid were placed in a round-bottom flask
at a molar ratio of 7:2:1 and were dissolved in
CHCl3-methanol (85:15; vol/vol). The organic solvents were
evaporated to dryness in a rotary evaporator under an N2 atmosphere. The dry lipid film was swelled with
125I-labeled BSA or with the drug for 1 h at 37°C
followed by sonication for 30 s. This suspension was centrifuged
in a Beckman ultracentrifuge (100,000 × g) for 30 min.
The pellet was similarly washed twice and was finally suspended in 2 ml
of phosphate-buffered saline (PBS; pH 7.4).
Grafting of fMLP onto the liposomal surface.
The coupling
between the ---NH2 group of PE liposomes and the ---COOH
group of fMLP was done by using EDC1 by following the
published procedure (5). In brief, the required amount of
fMLP was added dropwise to the liposomal suspension, followed by the
dropwise addition of EDC1. The pH of the solution was
maintained at 4.3 to 4.5 with 0.1 N HCl. The reaction was allowed to
proceed for 2 h at room temperature. The suspension was then
layered over 7 ml of Ficoll-Paque and was centrifuged at 750 × g
for 20 min. The liposome suspension floating on top was taken out
and was washed twice by centrifugation at (100,000 × g) for 30 min. The pellet finally obtained was
suspended in PBS containing 0.1 N CaCl2. The grafting
efficiency was about 50 to 60% with the various amounts of fMLP (see
Fig. 1).
In vitro uptake of peptide-grafted liposomes by
macrophages as a function of both peptide concentration and
time.
For the uptake study, the fMLP-grafted liposomes containing
125I-labeled BSA were used. A total of 200 µl of each
liposomal suspension (2 mg of phospholipid) in which various amounts of
fMLP (0 to 150 nmol) were grafted onto the liposomal surface was
incubated with the macrophages (105 cells in 300 µl of RPMI 1640 medium) at 37°C. After 1 h the suspension was
centrifuged (500 × g for 2 min) and washed twice and
the pellet was taken for counting of the radioactivity.
For studying the dependence of uptake with time, 200 µl of liposomal
suspension, both regular and modified with 100 nmol of
fMLP grafted
onto the liposomal surface, was incubated with the
cells
(10
5 in 300 µl of RPMI 1640 medium) at 37°C for various
time periods.
At different time intervals, the suspension was
centrifuged as
described above and washed and the radioactivity was
counted.
The nonspecific uptake study was done by preincubating the
macrophages
(10
5) with a large excess of free fMLP
(1 µmol) for 30 min at room
temperature. After 30 min, the cell
suspension was centrifuged
and washed. The cell pellet was suspended in
300 µl of RPMI 1640
medium. A total of 200 µl of the liposomal
suspension with 100
nmol of fMLP on the liposomal surface was added to
the cell suspension,
and the mixture was incubated at 37°C for
various time periods.
At various time intervals the suspension
was centrifuged and washed
and the radioactivity was counted. The
ungrafted liposomes were
used as controls. The cell protein in
all the cases was assayed
by the method of Lowry et al.
(
11).
In vitro parasite killing by the liposomes, free peptide, and
primaquine.
The macrophages (105 cells) were
incubated with L. donovani promastigotes at a ratio of 1:20
for 2 h at 37°C on a coverslip. After 2 h the nonadhering
cells were removed by washing with PBS. These infected
macrophages were then incubated with either 200 µl of free
peptide (100 nmol), 200 µl of empty liposomes, or 200 µl of peptide
(100 nmol)-grafted empty liposomes in RPMI 1640 medium at 37°C for
various time periods. At various time periods, the cover glasses were
removed and their contents were washed with PBS, air dried, and stained
with Giemsa. For each glass the total number of parasites per 300 cells
was counted. The untreated infected macrophages were used as
controls. The infected macrophages on the cover glass were also
incubated with 200 µl of free primaquine in RPMI 1640 medium over a
certain range of concentrations, washed, stained with Giemsa, and
examined for morphology and internal parasites.
Encapsulation of primaquine within the liposomes.
The
liposomes were prepared as described above. A certain amount of
primaquine was given during swelling of the lipid film. After 1 h,
the suspension was sonicated briefly, centrifuged at (100,000 × g for 30 min), and washed twice. All the washings were collected, and the amount of primaquine present in the supernatant was
determined by measuring the optical density at 360 nm (
= 3,100 M1 cm1). The level of encapsulation was
found to be 14%.
Animal experiment.
Our colony of golden hamsters
(Mesocricatus auratus), originally from the Haffkine
Research Institute, Bombay, India, was used to maintain L. donovani AG83 from an Indian patient with kala-azar by
intracardial passage every 6 weeks. Amastigotes were isolated from
spleen by the standard method (12). Each animal was injected
with 2 × 106 amastigotes intracardially. A group of
24 hamsters of average body weight (100 g) was infected at a time and
was ready for drug testing after 30 days. The hamsters (four in each
group) were distributed for drug treatment in the following manner: (i)
free primaquine (100 mg/kg of body weight), (ii) empty normal
liposomes, (iii) empty peptide-grafted liposomes, (iv) primaquine
encapsulated in ordinary liposomes, (v) primaquine encapsulated in
peptide-grafted liposomes, and (vi) untreated infected controls.
The amount of primaquine given to each animal was 100 mg/kg of body
weight. For determination of the 50% lethal dose, the
single dose
subcutaneous treatment was followed by treatment with
various amounts
of primaquine (40 to 500 mg/kg). The optimum dose
was found to be about
100 mg/kg of body weight. For chemotherapy,
a multiple-dose treatment
was used. In practice, 10 mg of primaquine
in 0.5 ml of liposomal
suspension containing 6 mg of lipid was
injected subcutaneously into
each hamster every 3 days for a total
of four doses over 10 days. Free
drug (10 mg/0.5 ml of PBS) was
also injected similarly. The animals
were killed 3 days after
the last injection. Parasite burdens were
assessed from stained
impression smears by using the formula of Stauber
et al. (
20).
The peptide concentration was kept constant (10 µmol/ml) for all
liposomal preparations used for the in vivo
experiments.
Investigation of drug toxicity.
A few parameters like blood
pathology, tissue histology, and specific enzyme levels related to
normal liver function were chosen to determine the toxic effects of the
drugs delivered in both the free and the liposomal forms. The animals
were killed after the drug treatment. The spleens of the animals were
removed for histological examination by eosin and hematoxylin
staining (9). Blood samples from the animals were assayed
for (i) levels of specific enzymes (e.g., serum glutamate
pyruvate transaminase [SGPT] and alkaline phosphatase
[4]), (ii) the serum bilirubin concentration
(17), (iii) blood urea concentration (14), and (iv) hemoglobin concentration by established procedures.
| |
RESULTS |
Uptake of peptide-grafted liposomes as a function of peptide
concentration and time.
The saturability of the peptide receptors
on the macrophage surface is shown by incubating the
macrophages with increasing amounts of peptide grafted onto the
liposomal surface at 37°C. The saturation occurred with about 100 nmol of peptide grafted onto the liposome surface (Fig.
1). The phagocytosis of fMLP-grafted liposomes by macrophages was time dependent and reached
equilibrium at about 30 min. The uptake at 4°C is very low and is
independent of time (data not shown). The process of binding between
the peptide receptors and peptide-grafted liposomes was found to be
specific in nature. Preincubation of the macrophages with
excess free fMLP inhibited further binding, followed by the
internalization of fMLP-grafted liposomes through the peptide
receptors. A nonsignificant decrease in uptake was noticed for
fMLP-grafted liposomes in comparison to that for the regular liposomes
(Fig. 2). The microbicidal activities of
fMLP itself and fMLP-grafted empty liposomes were determined by
incubating them separately with L. donovani-infected
macrophages for various time periods. The number of parasites
within the macrophages was markedly reduced within 3 h of
incubation compared to the number of parasites within untreated cells.
The almost similar extent of parasite killing for both the peptide and
the peptide-grafted liposomes indicated unaltered microbicidal activity
even after chemical modification. Parasite killing to the extent of 5 to 8% was also noticed for empty ungrafted liposomes (Table
1).
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FIG. 1.
Uptake of peptide-grafted liposomes by
macrophages as a function of peptide concentration. Results are
expressed as means ± standard deviations (n = 3).
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FIG. 2.
Specific uptake of peptide-grafted liposomes by
macrophages. Results are expressed as means ± standard
deviations (n = 3).
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|
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TABLE 1.
Microbicidal effect of free peptide, empty liposomes, and
peptide-grafted empty liposomes on L. donovani-infected
macrophages as a function of time (in vitro)
|
|
Microbicidal activity and toxicity of peptide-grafted liposomal
primaquine.
The microbicidal activity of fMLP-grafted
liposomal primaquine in the hamster model of a 30-day
L. donovani infection is shown in Table
2. About 14% of the drug used for
swelling was encapsulated within the liposomes 
max = 360 nm; = 3,100 M1 cm1). With multiple
treatments with free drug at a dose of 100 mg/kg of body weight, the
reduction in the spleen parasite load was 17.2%, whereas with the same
type of treatment but with the liposomal drug the reduction in the
spleen parasite load was 50%. The peptide-grafted empty liposomes and
the peptide-grafted liposomal primaquine reduced the spleen parasite
load by 31 and 75%, respectively. The reduction in the spleen parasite
load (10 to 15%) brought about by empty liposomes was probably the
result of an adjuvant effect.
In an attempt to determine the toxicity of the drug and/or the delivery
system itself, the levels of two specific enzymes
(alkaline phosphatase
and SGPT), serum bilirubin levels, and blood
urea levels were
determined. The results are presented in Table
3. The activities of both enzymes
increased with free drug treatment
but were reduced and became close to
the normal level with treatment
with peptide-grafted liposomal drug.
The serum bilirubin level
was maximum for the infected animal,
indicating liver blockage
by the parasites, but the level decreased
with the reduction in
the parasite load with treatment with the
peptide-grafted liposomal
drug. The almost constant level of urea in
blood indicated the
absence of any nephrotoxicity. The other blood
parameters like
the erythrocyte level, the leukocyte level, and the
hemoglobin
content were practically unchanged (data not shown).
Histological
examination of spleen was also performed after the various
treatments.
The sizes or shapes of the white pulp, red pulp, and
arteries
after the treatment of the fMLP-grafted liposomal drug were
very
close to those of a normal spleen (data not shown), again
indicating
reduced toxicity under the given conditions.
| |
DISCUSSION |
The reversibility of the binding (at 4°C) between the ligand and
the fMLP receptor on the cell surface has already been reported (19), but the internalization of the receptor-ligand complex leads to occasional irreversibility at 37°C. It has been reported that these receptors are not recycled on the cell surface during 2 h of incubation (16). The uptake in vitro of the
fMLP-grafted liposomes at 37°C is rapid and highly specific. The
uptake attained equilibrium in about 30 min. This in vitro specificity
of the receptor-ligand interaction was also noticed when it was tested in vivo. The peptide-grafted liposomes are found to be rapidly cleared
from the blood circulation (half-life, ~2 min) and taken up by the
cells of the reticuloendothelial system. It is known that neutrophils
possess the receptor for the peptides (2, 3, 16), but due to
the short circulation time, the level of uptake of these liposomes by
neutrophils is very low (~1%) compared to that of cells of the
reticuloendothelial system.
The macrophage-activating property of fMLP is well known,
resulting in the production of a series of reactive toxic oxygen metabolites like OH·, O2, and
H2O2 (2, 3). These metabolites are
the causative agent of nonspecific killing of various pathogens. The in
vitro time-dependent killing of the leishmania parasite within the
macrophages demonstrates that the activating property of fMLP
is not affected after the chemical grafting with the ---NH2
group of PE liposomes (Table 1).
The applicability of this delivery system has been tested in vivo
against experimental leishmaniasis in the hamster model by using
primaquine. The mechanism of action of primaquine is through the
production of O2 (13, 15). It has been
found that with equivalent drug concentrations, fMLP-grafted liposomes
are much more effective than either regular liposomes or free drug.
Interestingly, the empty fMLP-grafted liposomes reduced the spleen
parasite load by 31%, supporting the supposition that the
peptide-triggered production of O2 kills the
microorganisms. Therefore, both the drug alone and the delivery system
alone are capable of killing parasites through O2
production. Enhanced drug activity (Table 2) or reduced drug toxicity
(Table 3) is evidenced in the liposomal or the peptide-grafted liposomal form.
This study demonstrates that the efficacy of a drug against reversible
leishmaniasis is increased if the drug is used in the fMLP-grafted
liposomal form. Unlike other delivery systems, the fMLP-grafted
liposome is not only capable of delivering drugs to the
macrophages but it also activates the macrophage's
respiratory burst, leading to nonspecific killing of pathogens.
It appears that fMLP-grafted or other chemoattractant-grafted (1,
7) liposomes would possibly have useful applications against
other macrophage-associated disorders in the near future.
| |
ACKNOWLEDGMENTS |
Financial assistance from the Council of Scientific & Industrial
Research, New Delhi, India, and the Department of Science & Technology,
New Delhi, India, in the form of Senior Research Fellowship (to G.B.)
and Research Associateship (to S.M.) is gratefully acknowledged.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Biomembrane
Division, Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Rd., Calcutta 700 032, India. Phone: 91-033-4736795. Fax:
91-033-4735197. E-mail: iicb%sirnetc{at}sirnetd.ernet.in.
| |
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Antimicrobial Agents and Chemotherapy, February 1998, p. 348-351, Vol. 42, No. 2
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
