Interaction of acyclovir and its squalenoyl-acyclovir prodrug with DMPC in monolayers at the air/water interface

Article abstract of DOI:10.1016/j.ijpharm.2010.05.035

Acyclovir has been conjugated to the acyclic isoprenoid chain of squalene to form the squalenoyl-acyclovir prodrug. Its interaction with biomembrane models constituted by dimyristoylphosphatidylcholine (DMPC) monolayers has been studied by employing the Langmuir-Blodgett technique. The aim of the work was to gain information on the interaction of these compounds with phospholipid membranes. DMPC/acyclovir or squalenoyl-acyclovir prodrug mixed monolayers have been prepared at increasing molar fractions of the compound and the isotherm mean molecular area/surface pressure has been registered at 10 and 37 °C. Results reveal that the squalenoyl moiety enhances the affinity of acyclovir for the biomembrane model.

Full text of DOI:10.1016/j.ijpharm.2010.05.035

International Journal of Pharmaceutics 395 (2010) 167–173
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
Interaction of acyclovir and its squalenoyl–acyclovir prodrug with DMPC in
monolayers at the air/water interface
Maria Grazia Sarpietroa, Flavio Roccob, Dorotea Micielia, Sara Ottimoa,
Maurizio Cerutib, Francesco Castellia,∗
a Dipartimento di Scienze Chimiche, Università degli Studi di Catania, Viale Andrea Doria 6, 95125 Catania, Italy
b Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, Via Pietro Giuria 9, 10125 Torino, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Acyclovir has been conjugated to the acyclic isoprenoid chain of squalene to form the
squalenoyl–acyclovir prodrug. Its interaction with biomembrane models constituted by dimyristoylphos-
phatidylcholine (DMPC) monolayers has been studied by employing the Langmuir–Blodgett technique.
The aim of the work was to gain information on the interaction of these compounds with phospholipid
membranes.
Received 24 February 2010
Received in revised form 12 May 2010
Accepted 19 May 2010
Available online 26 May 2010
DMPC/acyclovir or squalenoyl–acyclovir prodrug mixed monolayers have been prepared at increasing
molar fractions of the compound and the isotherm mean molecular area/surface pressure has been reg-
istered at 10 and 37 ◦C. Results reveal that the squalenoyl moiety enhances the affinity of acyclovir for
the biomembrane model.
Keywords:
Acyclovir
Lipophilic acyclovir derivative
Dimyristoylphosphatidylcholine
Langmuir–Blodgett
Biomembrane models
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
eters, such as lipid composition, subphase properties, pH and
temperature can be chosen to imitate real biological conditions
Acyclovir [9-(2-hydroxyethoxymethyl)guanine] is a potent and
highly selective inhibitor of the replication of herpes viruses includ-
ing herpes simplex virus type 1 (HSV-I) and type 2 (HSV-2),
varicella-zoster virus, and Epstein-Barr virus both in cell cultures
and in animals. It is currently used as a therapeutic agent for
the treatment of these viruses’ infections (O’Brien and Campoli-
Richards, 1989; Wagstaff et al., 1994; Kim et al., 1998; Périgaud
et al., 1999). To exert its action acyclovir has to cross the cellu-
lar membrane and reach the intracellular compartment where the
viruses are hosted. A useful approach to improve this transfer pro-
cess could be the use of a lipophilic membrane soluble prodrug of
acyclovir, and/or the inclusion of the prodrug into a carrier prepa-
ration such as liposomes. Then, the knowledge of the interaction
between this prodrug and the biological membrane should be of
great importance.
(Kaganer et al., 1999; Brezesinski and Möhwald, 2003; Gaboriaud
et al., 2005). Moreover, Langmuir monolayers offer the possibil-
ity of controlling the molecular organization in a bi-dimensional
structure similar to that of the biological membranes (Maget-Dana,
1999; Dynarowicz-Latka et al., 2001). By the film-balance method
the surface pressure/mean molecular area (ꢀ/Å2) isotherm curves
are easily obtained. The phospholipids are spread on an aqueous
subphase to permit a monomolecular distribution. If the monolayer
is subjected to an area compression, the molecules distribution is
modified and the molecules are forced to go from a “gaseous” or
“liquid expanded” phase at low density to a “liquid condensed”
phase at a higher density and, successively, to a “solid condensed”
phase (Gaines, 1966; Mohwald, 1990; Prieto et al., 1998; Krasteva
et al., 2000; Vollhardt and Fainerman, 2000; Broniec et al., 2007).
Eventual variations in the behavior of the isotherms of the pure
phospholipid, caused by the presence of a compound dispersed
among the phospholipids on the aqueous surface can indicate
anomalies in the perfect miscibility of the phospholipid/compound
mixtures.
The lipid membranes are of highly complex structures, hence
their biophysical interactions with drugs are difficult to investigate.
Simplified artificial membranes systems mimicking the natural
lipid membranes have been developed and used to study these
interactions. Among the various models of cell membranes, lipid
Langmuir monolayers are extensively used because several param-
In previous studies of some of us, this technique was applied
to define the localization of lipophilic prodrugs of gemcitabine
(Stella et al., 2004/2005) in phospholipids monolayers (Castelli
et al., 2007a,b). In the present study, the interaction between a
lipophilic acyclovir prodrug (squalenoyl–acyclovir), obtained by
conjugation of acyclovir with the squalene acyclic isoprenoid chain
∗
Corresponding author. Tel.: +39 095 221796; fax: +39 095 580138.
E-mail address: fcastelli@dipchi.unict.it (F. Castelli).
0378-5173/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijpharm.2010.05.035

168
M.G. Sarpietro et al. / International Journal of Pharmaceutics 395 (2010) 167–173
(2.3 ml) was added at 0 ◦C to distilled water (20 ml) with stir-
ring, followed by potassium dichromate (1.21 g, 4.11 mmol) to
obtain chromic acid. It was then added at 0 ◦C within 20 min to
the solution of the aldehyde previously prepared and left to react
for 2 h at 0 ◦C with stirring. The reaction mixture was extracted
with diethyl ether (50 ml 3×), washed with saturated brine, dried
over anhydrous sodium sulfate and evaporated in vacuo. The com-
pletion of the reaction was revealed by silica gel TLC with light
petroleum/diethyl ether/methanol, 70:23:7. The crude product was
purified by flash chromatography with light petroleum, then light
petroleum/diethyl ether, 95:5 as eluant, to give 577 mg of 1,1ꢀ,2-
tris-nor-squalene acid (35% yield), as a colourless oil. 1H NMR
(CDCl3): ı, 1.55–1.63 (m, 18H, allylic CH3), 1.90–2.05 (m, 16H,
allylic CH2), 2.26 (t, 2H, CH2CH2COOH), 2.38 (t, 2H, CH2CH2COOH),
5.00–5.19 (m, 5H, vinylic CH), 12.20 (broad, 1H, COOH). MS (EI):
m/z 400 (M+, 5), 357 (3), 331 (5), 289 (3), 208 (6), 136 (3), 81 (100).
2.2.2. Squalenoyl–acyclovir: 9-[-(4E,8E,12E,16E)-4,8,13,17,21-
pentamethyl-4,8,12,16,20-docosapentaenoyloxyethoxymethyl]-2-
amino-1,7-dihydro-6H-purin-6-one
1,1ꢀ,2-Tris-nor-squalene acid (178 mg, 0.444 mmol) was dis-
solved in anhydrous DMF (1.5 ml) in a three-necked flask under
nitrogen, with stirring, at room temperature, followed by dimethy-
laminopiridine chloridrate (DMAP) (×2.2; 120 mg, 0.98 mmol) in
anhydrous DMF (1.5 ml). Acyclovir (100 mg, 0.444 mmol) was dis-
solved in anhydrous DMF (2 ml), with soft heating and slowly added
to the previously prepared solution heated at 60 ◦C in a silicon
bath, with stirring. When the reaction mixture resulted homoge-
neous, N-(3-dimethylaminopropyl)-Nꢀ-ethylcarbodiimide (EDCA)
(×2.2; 188 mg, 0.98 mmol), dissolved in anhydrous DMF (1.5 ml)
was added and allowed to react for 3 days, with stirring, under
nitrogen, at 70 ◦C. The reaction mixture was controlled by silica gel
TLC with ethanol/chloroform/cyclohexane, 50:25:25, transferred in
a one-necked flask and evaporated to dryness. The crude deriva-
tive was dissolved in dichloromethane, washed with a 3% aqueous
HCl solution, washed with saturated brine until neutral pH, dried
over anhydrous sodium sulfate and evaporated in vacuo. The crude
product was purified by silica gel flash chromatography previously
eluded alone with dichloromethane/triethylamine, 99:1 and neu-
tralized with dicloromethane. The elution of the crude product was
performed with dichloromethane/ethanol, 95:5 to give 115 mg of
squalenoyl–acyclovir (42% yield), as a pale yellow viscous oil. It
was completely pure, as revealed by 1H NMR and mass analysis.
Concerning the possibility of obtaining either an ester or an amide
linkage between the squalenoyl chain and acyclovir, 1H NMR anal-
ysis revealed the presence of the free amino group at 6.57 ı (2H, s,
NH2), while the signal at about 12 ı of the hypothetic amidic group
was completely absent. It was also confirmed by comparison of
1H NMR spectra of ester and amide acetyl derivatives of acyclovir
reported in literature (Matsumoto et al., 1987). 1H NMR (DMSO): ı,
1.52–1.65 (18H, m, allylic CH3), 1.94–2.10 (16H, m, allylic CH2), 2.13
(2H, t, OCOCH2CH2), 2.31 (2H, t, OCOCH2), 3.63 (2H, m, 3ꢀ-OCH2),
4.06 (2H, m, 4ꢀ-CH2OCO), 5.03–5.25 (5H, m, vinylic CH), 5.32 (2H,
s, 1ꢀ-NCH2O), 6.57 (2H, s, NH2), 7.78 (1H, s, 8-CH), 10.69 (1H, s,
1-NHCO). MS (CI): m/z 608 (M+, 100).
Scheme 1. Acyclovir, squaleneCOOH and squalenoyl–acyclovir structure.
(squaleneCOOH) (Scheme 1), and dimyristoylphosphatidylcholine
(DMPC) in mixed monolayers was explored. To have more infor-
mation, the interaction of the non-conjugate compounds (acyclovir
and squaleneCOOH) with DMPC was also investigated. Results per-
mit to obtain indication on the ability of the examined compounds
to dissolve, or not, in the phospholipid molecules that form the
monolayer and then to have indication on their interaction with
phospholipids membranes.
2. Materials and methods
2.1. Materials
Acyclovir (purity ≥ 99%) and squalene (purity = 98%) were
purchased from Sigma–Aldrich (Italy). 1,1ꢀ,2-Tris-nor-squalene
aldehyde was obtained from squalene as previously described
(Ceruti et al., 2005). Synthetic l-␣-dimyristoylphosphatidylcholine
(DMPC) (purity ≥ 98%) was obtained from Genzyme (Switzer-
land). Lipids were chromatographically pure as assessed by
two-dimensional thin layer chromatography.
2.3. Surface tension measurements
Film-balance measurements were performed using a KSV mini-
trough apparatus provided with a computer interface unit and
an operating software. The trough (24,225 mm2 available area)
made in Teflon was connected to a circulating water bath to
keep the temperature constant. 5 mM Tris (pH = 7.4) in ultra-
pure Millipore water with resistivity of 18.2 Mꢁ cm was used
as subphase. Equimolar solutions (0.001 mmol/ml) of DMPC,
squaleneCOOH, squalenoyl–acyclovir and acyclovir in organic sol-
2.2. Synthesis of the compounds
2.2.1. 1,1ꢀ,2-Tris-nor-squalene acid: (4E,8E,12E,16E)-
4,8,13,17,21-pentamethyl-4,8,12,16,20-docosapentaenoic
acid
1,1ꢀ,2-Tris-nor-squalene aldehyde (1.58 g, 4.11 mmol) was dis-
solved in diethyl ether (20 ml) at 0 ◦C. Separately, sulfuric acid

M.G. Sarpietro et al. / International Journal of Pharmaceutics 395 (2010) 167–173
169
vents were prepared. Mixed DMPC/compound solutions were
successively prepared to obtain the following molar fractions for
each compound: 0.015, 0.03, 0.045, 0.06, 0.09, 0.12, 0.25, 0.50, and
0.75. 30 ␮l of the mixed solutions as well as the pure components
were spread drop-by-drop on the surface of the subphase by a
Hamilton microsyringe (which, before use, was cleaned three times
with chloroform and the examined solution) and, after waiting
15 min for solvent evaporation, the monolayers were compressed
by the use of two mobile barriers made in Delrin at the constant
speed of 10 mm/min. Surface pressure vs. molecular area isotherms
were recorded by the Wilhelmy plate arrangement attached to
a microbalance. Before spreading the sample, the subphase was
checked twice by running blank experiments to be sure that no
impurities were present. The experiments were performed at a sub-
phase temperature of 10 and 37 ◦C. Each experiment was repeated
at least three times to be sure of the reproducibility of the isotherm
measurements.
2.4. Surface pressure/molecular area isotherms analysis
2.4.1. Monolayer miscibility
The surface pressure/molecular area isotherms were analyzed
by calculating, at different surface pressures, the molecular area
as a function of the monolayer composition (expressed in molar
fraction). The averaged molecular area of a two-components mono-
layer can be calculated by A = A1X1 + (1 − X1) A2, where A is the mean
molecular area, X1 is the molar fraction of component 1, X2 = 1 − X1,
and A1 and A2 are the areas per molecule of the monolayer pure
components at the same surface pressure. When the two compo-
nents are either ideally miscible or completely immiscible at the
air/liquid interface, reporting in a graph A as a function of X1, a
straight line is obtained (Gaines, 1966; Shahgaldian and Coleman,
2003). Any deviation from the straight line indicates that the mis-
cibility of the components is non-ideal.
2.4.2. Monolayer stability
A convenient and powerful tool to evaluate mixed monolayer
stability is excess Gibb’s energy (ꢂGex). In case of ideal mixing
between monolayer components, where one component is com-
pletely mixed with the other monolayer component, ꢂGex attains
0 value. Similarly, the presence of repulsive interactions between
monolayer components results in a positive Gibb’s excess, while
associative or attractive interactions result in a negative Gibb’s
excess (Chou and Chang, 2000). Excess free energy of mixing
ꢀ
was calculated by applying the equation: ꢂGex ꢀ[A12 − (X1A1
+
X2A2)] dꢀ; where X1 and X2 are the molar fraction0s of the two com-
ponents, while A1 and A2 are the molar areas of the monolayer
pure components; A12 is the effective molar area occupied by the
mixed monolayer and ꢀ is the surface pressure. Positive Gibb’s
excess is suggestive of the formation of thermodynamically unsta-
ble monolayers, while a negative Gibb’s excess indicates stable
systems (Chimote and Banerjee, 2008).
Fig. 1. Surface pressure/molecular area isotherms of DMPC and (A) acyclovir, (B)
squaleneCOOH and (C) squalenoyl–acyclovir mixed monolayers at the air–water
interface at 10 ◦C.
sure and temperature. At low pressure, the DMPC monolayer is in a
gaseous-like phase (the surface area per molecule ranging from 120
to about 100 Å2) with the acyl chains most apart in the air. Further
compression forces the monolayer into the liquid expanded (LE)
phase (area between 100 and 65 Å2); then the monolayer is sub-
jected to a liquid-expanded–liquid-condensed (LE–LC) transition
(area from about 65 to 45 Å2). Finally, a further compression yields
a liquid condensed (LC) phase (area lower than 45 Å2) producing a
steep rise in the surface tension.
Acyclovir does not form monolayers, but its presence, at molar
fraction higher than 0.09, causes the isotherms to gradually move
towards lower molecular areas and the LE–LC transition tempera-
ture to gradually decrease (Fig. 1A). SqualeneCOOH is in a gaseous
state in the range of 120–85 Å2, and in a LE state at areas smaller
than 85 Å2. This compound, at low molar fraction, does not cause
3. Results and discussion
3.1. Molecular area/surface pressure isotherms
Mixed monolayers of DMPC/acyclovir, DMPC/squaleneCOOH
and DMPC/squalenoyl–acyclovir at the air/water interface have
been prepared and their behavior has been studied and com-
pared with that of single component monolayers. The molecular
area/surface pressure isotherms have been recorded at 10 ◦C
(Fig. 1A–C) and 37 ◦C (Fig. 2A–C), below and above the pure phos-
pholipid transition temperature.
10 ◦C measurements. When distributed as
molecules exist in different states, depending upon surface pres-
a monolayer,

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M.G. Sarpietro et al. / International Journal of Pharmaceutics 395 (2010) 167–173
37 ◦C measurements. DMPC shows two regions: a gaseous phase
from 120 to 110 Å2 and a LE region starting from 110 Å2. Even at this
temperature, acyclovir does not show any isotherm. Up to 0.03 M
fraction, acyclovir causes the isotherm to move towards higher
molecular area, but at higher molar fraction, it causes the isotherm
to move towards lower molecular area (Fig. 2A). SqualeneCOOH is
Fig. 2. Surface pressure/molecular area isotherms of DMPC and (A) acyclovir, (B)
squaleneCOOH and (C) squalenoyl–acyclovir mixed monolayers at the air–water
interface at 37 ◦C.
significant variations of the isotherm: at 0.25 M fraction the LE–LC
transition disappears and the isotherm moves towards higher
value of the molecular area; higher squaleneCOOH molar fractions
cause the isotherms to shift towards lower molecular area values
(Fig. 1B).
Squalenoyl–acyclovir is characterized by a gaseous phase from
120 to 65 Å2, a LE phase from 65 to about 35 Å2, a LE–LC transi-
tion from about 35 to 10 Å2, and then a LC phase. The presence
of squalenoyl–acyclovir, up to 0.09 M fraction, does not determine
substantial variations of the DMPC isotherms, just a small shift
towards higher molecular areas; at 0.12 M fraction, the isotherms
lose the LE–LC transition, although their shape remains almost
unchanged. As the squalenoyl–acyclovir molar fraction further
increases, the isotherms move towards lower molecular areas and
exhibit a well-defined transition (Fig. 1C).
Fig. 3. Molecular area of the mixed monolayers of DMPC and (A) acyclovir, (B)
squaleneCOOH and (C) squalenoyl–acyclovir at the air–water interface plotted as
a function of the molar fraction of compound at various values of surface pressures
at 10 ◦C.

M.G. Sarpietro et al. / International Journal of Pharmaceutics 395 (2010) 167–173
171
in a gaseous state at molecular areas between 120 and 60 Å2, and
3.2. Surface pressure/molecular area isotherms analysis
3.2.1. Monolayer miscibility
The mean molecular area of the mixed monolayers has been
reported as a function of compound molar fraction at 10, 20 and
30 mN/m (Figs. 3A–C and 4A–C).
in a LE state at lower areas. The increase of squaleneCOOH molar
fraction in DMPC monolayers determines the isotherm to gradually
shift towards smaller areas (Fig. 2B). Even at 37 ◦C, as the com-
pression increases, squalenoyl–acyclovir presents four regions: a
gaseous phase from 120 to 60 Å2, a LE phase from 60 to about
30 Å2, a LE–LC transition from about 30 to 15 Å2, and a LC phase
for smaller molecular areas. No significant variations are noted in
the isotherms with squalenoyl–acyclovir up to 0.12 M fraction; at
higher molar fractions, squalenoyl–acyclovir, causes the isotherms
to shift towards lower molecular areas and the LE–LC transition to
appear and became more evident (Fig. 2C)
Fig. 4. Molecular area of the mixed monolayers of DMPC and (A) acyclovir, (B)
squaleneCOOH and (C) squalenoyl–acyclovir at the air–water interface plotted as
a function of the molar fraction of compound at various values of surface pressures
at 37 ◦C.
Fig. 5. Excess Gibb’s free energies of mixing in DMPC and (A) acyclovir, (B)
squaleneCOOH and (C) squalenoyl–acyclovir mixed monolayer as a function of the
molar fraction of compound at various values of surface pressures at 10 ◦C.

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M.G. Sarpietro et al. / International Journal of Pharmaceutics 395 (2010) 167–173
10 ◦C measurements. With regard to acyclovir/DMPC mixed
squaleneCOOH molar fractions, the experimental values overlap
the ideal behavior, suggesting a complete and the ideal miscibility
between the monolayer components. At high squaleneCOOH molar
fractions, positive deviations are present indicating that repulsive
interactions occur among squaleneCOOH and DMPC molecules. The
behavior of squalenoyl–acyclovir/DMPC mixed monolayers pos-
itively deviate from the ideality at all the squalenoyl–acyclovir
molar fractions and surface pressures, suggesting repulsive inter-
actions between the monolayer components (Fig. 3C).
monolayers (Fig. 3B), at low molar fraction of acyclovir, the exper-
imental values almost coincide with the ideal values, suggesting
good miscibility between acyclovir and DMPC; at high acyclovir
molar fraction, a positive deviation is found, suggesting the
formation of a non-ideal monolayer with dominant repulsive inter-
actions. In squaleneCOOH/DMPC mixed monolayers (Fig. 3B), at low
37 ◦C measurements. Positive deviations, and hence repulsive
interactions, are observed in acyclovir/DMPC mixed monolayers
for all the acyclovir molar fractions and surface pressures, partic-
ularly at 20 mN/m (Fig. 4A). Increasing the squaleneCOOH molar
fraction, the squaleneCOOH/DMPC mixed monolayers exhibit first
negative deviations, then, positive deviations for all the consid-
ered surface pressures. It suggests the formation of immiscible
monolayers where attractive and repulsive interactions occur at
low and high squaleneCOOH molar fraction, respectively (Fig. 4B).
Squalenoyl–acyclovir mixed monolayers, up to 0.045 M fraction of
squalenoyl–acyclovir, show an ideal behavior. On increasing the
squalenoyl–acyclovir molar fraction, especially at 20 mN/m, the
occurrence of positive deviations is suggestive of repulsive forces
between DMPC and squalenoyl–acyclovir (Fig. 4C).
3.2.2. Monolayer stability
10 ◦C measurements. The behavior of ꢂGex against the acyclovir
molar fraction is shown in Fig. 5A. On increasing the acyclovir
concentration, negative and positive deviations of ꢂGex alter-
nate, suggesting stabilization and de-stabilization of the monolayer
with acyclovir concentration. With regard to squaleneCOOH
(Fig. 5B), generally positive deviations are found, indicating the
formation of squaleneCOOH/DMPC unstable monolayers. In the
case of squalenoyl–acyclovir (Fig. 5C), at all prodrug molar
fractions, positive deviations are present suggesting unstable
squalenoyl–acyclovir/DMPC monolayers.
37 ◦C measurements. Acyclovir (Fig. 6A) causes positive devi-
ation in the acyclovir/DMPC monolayer, especially at low molar
fractions, leading to the formation of unstable monolayers. At low
molar fraction, squaleneCOOH (Fig. 6B) causes negative deviations,
becoming positive at high molar fractions. This suggests that at
low molar fraction of compound, a stable monolayer exists, which
becomes unstable by increasing the squaleneCOOH molar fraction.
In the squalenoyl–acyclovir/DMPC monolayers (Fig. 6C), the exper-
imental values overlap the ideal values at very low molar fractions
of squalenoyl–acyclovir, then strong positive deviations (unstable
monolayers) are observed.
4. Conclusion
The lipophilic squalenoyl–acyclovir prodrug was obtained by
conjugation of acyclovir with the squalene acyclic isoprenoid chain.
The behavior of the prodrug, acyclovir and squaleneCOOH in the
monolayer at the air/water interface and their interaction with a
biomembrane model made up of dimyristoylphosphatidylcholine
monolayers was studied. The above reported Langmuir–Blodgett
measurements give information not obtainable by other tech-
niques, evidencing that the squaleneCOOH group confers to the
prodrug a behavior quite different than that shown by acyclovir.
In particular, acyclovir does not form any monolayer, while the
prodrug forms a stable monolayer which evidence a characteris-
tic phase transition upon compression. This transition is preserved
in DMPC/squalenoyl–acyclovir mixed monolayers at high prodrug
molar fractions. All the studied compounds interact with DMPC,
as also reported in previous studies carried out by differential
scanning calorimetry of mixed multilamellar vesicles (Sarpietro
Fig. 6. Excess Gibb’s free energies of mixing in DMPC and (A) acyclovir, (B)
squaleneCOOH and (C) squalenoyl–acyclovir mixed monolayer as a function of the
molar fraction of compound at various values of surface pressures at 37 ◦C.

M.G. Sarpietro et al. / International Journal of Pharmaceutics 395 (2010) 167–173
173
et al., 2009). The different effect of the investigated compounds
on DMPC monolayer is considerably influenced by the presence
of the squalenoyl group bound to acyclovir which modifies the
shape of the isotherms. These effects are much more evident than
those previously observed by calorimetry (Sarpietro et al., 2009).
In particular, the positive deviations of both the surface area excess
(Figs. 3 and 4) and the Gibb’s free energy excess (Figs. 5 and 6)
clearly indicate repulsive interactions between the monolayer
components. Furthermore, the comparison of all the above results
may provide information on the localization of the three com-
pounds in DMPC monolayers. Acyclovir produces a small expansion
effect, probably because it is localized near the DMPC polar heads
and do not contribute to the hydrophobic interactions among the
phospholipid chains. SqualeneCOOH could be localized parallel
to the phospholipids chain with the carboxylic group protruding
towards the subphase. The increase of the lipophilic character of
acyclovir through its conjugation to squaleneCOOH and the forma-
tion of the squalenoyl–acyclovir prodrug gives rise to a stronger
interaction with DMPC monolayer with respect to those of the free
drug. This effect probably arises because the prodrug molecules is
inserted among the DMPC acylic chain in a way that the phospho-
lipids molecules are forced to occupy a larger area. Almost the ideal
behavior observed at 37 ◦C and at low molar fraction could depend
on the flexibility of the molecule at this temperature, which well
adapts to the surrounding phospholipid chains.
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Acknowledgments
We thank the MIUR (Fondi di Ateneo) and the Regional Govern-
ment (Regione Piemonte) for financial support.
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