Anomalous interaction of tri-acyl ester derivatives of uridine nucleoside with a l-α-dimyristoylphosphatidylcholine biomembrane model: a differential scanning calorimetry study

Article abstract of DOI:10.1111/jphp.13038

Objectives: Uridine was conjugated with fatty acids to improve the drug lipophilicity and the interaction with phospholipid bilayers. Methods: The esterification reaction using carbodiimides compounds as coupling agents and a nucleophilic catalyst allowed us to synthesize tri-acyl ester derivatives of uridine with fatty acids. Analysis of molecular interactions between these tri-acyl ester derivatives and l-α-dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles (MLV) – as a mammalian cell membrane model – have been performed by differential scanning calorimetry (DSC). Key findings: The DSC thermograms suggest that nucleoside and uridine triacetate softly interact with phospholipidic multilamellar vesicles which are predominantly located between the polar phase, whereas the tri-acyl ester derivatives with fatty acids (myristic and stearic acids) present a strongly interaction with the DMPC bilayer due to the nucleoside and aliphatic chains parts which are oriented towards the polar and lipophilic phases of the phospholipidic bilayer, respectively. However, the effects caused by the tri-myristoyl uridine and tri-stearoyl uridine are different. Conclusions: We show how the structural changes of uridine modulate the calorimetric behaviour of DMPC shedding light on their affinity with the phospholipidic biomembrane model.

Full text of DOI:10.1111/jphp.13038

Research Paper
Anomalous interaction of tri-acyl ester derivatives of uridine
nucleoside with a L-a-dimyristoylphosphatidylcholine
biomembrane model: a differential scanning calorimetry
study
a
a
a,b
ꢀ
ꢀ
ꢀ
Jhon Fernando Berrıo Escobar , Diana Margarita Marquez Fernandez , Cristiano Giordani
,
Francesco Castellic and Maria Grazia Sarpietroc
a
b
ꢀ
ꢀ
Productos Naturales Marinos, Departamento de Farmacia, Facultad de Ciencias Farmaceuticas y Alimentarias, Instituto de Fısica, Universidad de
c
ꢀ
ꢁ
Antioquia, UdeA, Medellın, Colombia and Dipartimento di Scienze del Farmaco, Universita degli Studi di Catania, Catania, Italia
Keywords
Abstract
anticancer nucleoside; biomembrane
models; differential scanning calorimetry;
fatty acid; uridine
Objectives Uridine was conjugated with fatty acids to improve the drug
lipophilicity and the interaction with phospholipid bilayers.
Methods The esterification reaction using carbodiimides compounds as coupling
agents and a nucleophilic catalyst allowed us to synthesize tri-acyl ester deriva-
tives of uridine with fatty acids. Analysis of molecular interactions between these
tri-acyl ester derivatives and L-a-dimyristoylphosphatidylcholine (DMPC) multil-
amellar vesicles (MLV) – as a mammalian cell membrane model – have been per-
formed by differential scanning calorimetry (DSC).
Correspondence
Maria Grazia Sarpietro, Dipartimento di
ꢁ
Scienze del Farmaco, Universita degli Studi
di Catania, Viale A. Doria 6, 95125 Catania,
Italy.
E-mail: mg.sarpietro@unict.it
Key findings The DSC thermograms suggest that nucleoside and uridine triac-
etate softly interact with phospholipidic multilamellar vesicles which are predom-
inantly located between the polar phase, whereas the tri-acyl ester derivatives
with fatty acids (myristic and stearic acids) present a strongly interaction with
the DMPC bilayer due to the nucleoside and aliphatic chains parts which are ori-
ented towards the polar and lipophilic phases of the phospholipidic bilayer,
respectively. However, the effects caused by the tri-myristoyl uridine and tri-
stearoyl uridine are different.
Received June 25, 2018
Accepted October 19, 2018
doi: 10.1111/jphp.13038
Conclusions We show how the structural changes of uridine modulate the
calorimetric behaviour of DMPC shedding light on their affinity with the phos-
pholipidic biomembrane model.
Anticancer nucleosides present numerous pharmacoki-
netic problems, such as low selectivity in attacking cancer
Introduction
The anticancer nucleoside and analogues drugs have been
widely used for cancer treatment since the 1960s, against
solid (e.g. lung, liver and breast) and soft (e.g. leukaemia
and non-Hodgkin lymphoma) tumours. They show cyto-
static, cytotoxic and antiproliferative activities due to their
action mechanisms as antimetabolites for the DNA (alpha
and epsilon polymerase inhibition) and RNA synthesis, and
for the purine and pyrimidine nucleosides synthesis. Their
incorporation into DNA chain causes demethylation and
hypomethylation of fragments. Moreover, some nucleoside
drugs can show activation of apoptosis routes on cytoplasm
and mitochondrial side.[1–6]
cells, and an extracellular inactivation and degradation due
to their high chemical and biological lability. They also
show very pharmacodynamics disadvantages, some of these
are as follows: low cellular uptake, intracellular inactivation
and biological degradation by means of ecto-nucleotidase
and nucleoside deaminase enzymes.[2,4–7] Due to the great
disadvantages revealed, the nucleoside drugs have a very
short plasma half-life and very low oral bioavailability;
therefore, these drugs type should be administered in
higher doses and intravenously to be effective. This admin-
istration form is responsible for severe side effects as gas-
trointestinal disorder, neurotoxicity, nephrotoxicity and
© 2018 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2018), pp. **–**
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Uridine prodrugs/DMPC bilayers interaction
Jhon Fernando Berrıo Escobar et al.
hepatotoxicity, among others, caused for the significant cell
death showed on healthy tissues adjacent to tumour or in
other organs.[2,4–8] Due to the disadvantages and adverse
effects, a substantial number of studies have been focused
on improving the biological and physico-chemical proper-
ties of nucleoside drugs, through the synthesis of aliphatic
derivatives with short and long aliphatic acids chains, aro-
matic derivatives and conjugates synthesized with hydroxyl
substances of big molecular weight as polymer and sterols.
These derivatives can provide protection against enzymatic
or chemical degradation and enhance the cellular uptake or
transport across cell membranes[2,4,7–11] Other investiga-
tions have been focused on the search of new nucleosides
or analogues that exhibit a better physico-chemical and/or
biological characteristic. It has been found that uridine, an
essential nucleoside, and some natural derivatives with
oligosaccharide or phosphate as substituents, among
others, show cytotoxic activities to diverse tumour cell lines
to higher concentrations than physiological levels of
these substances. Some of these compounds behave as
antimetabolites, interrupt the essential nucleoside and RNA
synthesis or act as inhibitors of glycosyltransferases
enzymes.[12–14] Other contributions based on the use of
synthetic derivatives of the same nucleoside, with aliphatic
substituent and/or halogen groups, ester with aliphatic
acids of long chains and aromatic acids, and conjugates
with triterpenoids hydroxylated, have showed significant
cell viability inhibition against solid and soft tumours cell
lines.[15–17] Therefore, in the oncologic therapy is highly
desired to obtain novel drugs or enhanced anticancer drugs
that exhibit improvement on pharmacological or biological
properties, to be used in chemotherapy.[18]
with biological membranes is not straightforward since cel-
lular membranes are chemically and physico-chemically
very complex.[19–23] Although very simplified compared to
the complexity of biological cell membranes, lipid bilayers
and monolayers can be employed as useful models. The
biomembrane models permitted to investigate the nature of
the interaction between phospholipids and drugs or com-
pounds with biological activity.[24–26] The L-a-dimyristoyl-
phosphatidylcholine (DMPC) is a saturated phospholipid
that is found in high quantities as component of healthy
and tumour cell membranes of mammalian. It easily builds
phospholipid structures as monolayer and liposomes, and
the main transitions are under of 37 °C, which is the human
corporal temperature; therefore, the DMPC is widely used
as phospholipid for the biomembrane models.[19,21,22]
The analysis of the diverse types of the interaction
involved between DMPC and drugs can be performed by
differential scanning calorimetry (DSC),[7,27] a technique
widely employed in several studies, because the thermody-
namic properties and technical parameters can be accu-
rately and easily monitored. The procedure for the
formation of liposomes is relativity simple and allows to
use phospholipids of several types as biomembrane models.
Moreover, this method is trustworthy in revealing details of
the diverse types of interactions of drugs, prodrugs and bio-
logically active substances of different polarity with diverse
phases of MLV; therefore, offering information about the
miscibility and distribution of the compounds analysed
over the phospholipid structures.[20,24,25]
Here, we prepared DMPC-MLV as a simplified model of
biomembrane and we studied, by DSC, the interactions
between uridine and the nucleoside derivatives and the
phospholipid bilayers.
It is noteworthy to point out that the tri-acyl ester
derivative of uridine with acetic acid, used in chemotherapy
in combination with antiviral (stavudine) and anticancer
(capecitabine) nucleoside drugs, has shown a decreasing of
the side effects (in terms of neurotoxicity showed by the
antiviral drug and gastrointestinal disorder and cardiotoxi-
city, presented by the anticancer nucleoside) exhibited by
these drugs when employed in high doses or administrated
for longer times, whereas the anticancer and antiviral activ-
ities remain unaltered. The uridine triacetate is a drug
(namely Vistogardâ) approved by The US Food and Drugs
Administration (FDA) and used in the treatment of over-
doses and numerous life-threatening toxicities from the
capecitabine anticancer nucleoside.[14,18]
Materials and Methods
Chemicals
All solvents employed were of reactive grade. The following
substances were used for the synthesis: uridine 99% (Alfae-
sar, Karlsruhe, Germany) as substrate; acetic anhydride,
myristic acid and stearic acid 99% (Merck, Darmstadt, Ger-
many) were employed as substituents; N,N0-diciclohexil-
carbodiimide (DCC) 99% (Alfaesar, Karlsruhe, Germany),
and 4-N,N-dimethylamino-pyridine (DMAP) 99%
(Alfaesar, Karlsruhe, Germany) as coupling agent and
nucleophilic catalyst, respectively. L-a-dimyristoylphospha-
tidylcholine (DMPC) 99% (Genzyme Pharmaceuticals,
Liestal, Switzerland) and tris-hydroximethyl-amino-
methane (TRIS) (Merck, Darmstadt, Germany) were
employed for the preparation of phospholipidic multilayer
vesicles (MLV). The reaction mixture was monitored by
TLC on F254 silica gel (Merck, Darmstadt, Germany), the
Studies on the interaction between cell membranes and
anticancer drugs (or prodrugs), are very interesting for sev-
eral reasons, including the understanding of the mechanism
of action, the miscibility process, the diffusion through the
phospholipidic bilayer structures, as well as the membrane
structural changes induced by the substances evaluated.
However, the analysis of interactions of drugs (or prodrugs)
2
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Jhon Fernando Berrıo Escobar et al.
Uridine prodrugs/DMPC bilayers interaction
products were purified by chromatography column normal
phase, some compounds were re-purified by preparative
TLC on normal phase, as reported elsewhere.[16,17]
(CDCl3), d ppm (multiplicity; integration; J (Hz); posi-
tion): 0.92 (t; 9H; J = 6.6; -CH3; H-14″ myristoyl
chain), 1.32 (m; 48H; H-4″ to H-13″ myristoyl chain),
1.70 (m; 6H; H-3″ (b protons) myristoyl chain), 2.40
(m; 6H; H-2″ (a protons) myristoyl chain), 4.38 (m;
3H; H-60 and H-50 ribose ring), 5.36 (m; 2H; H-30 and
H-40 ribose ring), 5.80 (d; 1H; J = 8.0; H-5 uracil ring),
6.08 (d, 1H, J = 5.1; H-20 ribose ring), 7.45 (d, 1H,
J = 8.1; H-6 uracil ring). 13C NMR spectroscopy data
and 2D correlations as reported elsewhere.[17,19] ESI-MS
(positive mode) m/z: Calculated for C51H90N2O9:
875.2683; found, 898.6 [M+Na]+.
30,40,60-O-tristearoyl uridine or uridine tri-stearate, was
isolated as a white solid (20% yield), melting point: 68–
69 °C. 1H NMR (CDCl3), d ppm (multiplicity; integra-
tion; J (Hz); position): 0.87 (t; 9H; J = 6.5; H-18″ stear-
oyl chain), 1.25 (m; 48H; H-4″ to H-17″ stearoyl acid),
1.56 (m; 6H; H-3″ (b protons) stearoyl acid), 2.32 (m;
6H; 2″ (a protons) stearoyl acid), 4.35 (m; 3H; H-60 and
H-50 ribose ring), 5.35 (m; 2H; H-30 and H-40 ribose
ring), 5.80 (d; 1H; J = 8.0; H-5 uracil ring), 6.05 (d, 1H,
J = 4.6; H-20 uracil ring), 7.49 (d, 1H, J = 8.2; H-6 uracil
ring). 13C NMR spectroscopy data and 2D correlations as
reported elsewhere.[17] ESI-MS (positive mode) m/z: Cal-
culated for C63H114N2O9: 1043.5872; found, 1066.9
[M+Na]+ and 1044.9 [M+H]+.
The tri-acyl ester derivatives of uridine isolated, were
characterized by: mass spectrometry, recorded on Agilent
6300-LC/MS Equipment (electrospray ionization (ESI) and
Ion-Trap analyzer) and an Agilent 6100 Series-LC/MS (ESI
and single quadrupole analyzer), both analyses in positive
mode and direct injection method, and nuclear magnetic
resonance spectroscopy (NMR) unidimensional and
bidimensional spectra, recorded on a Bruker 300 NMR
spectrometer, with tetramethylsilane (TMS) employed as
internal standard. The CDCl3 (99% deuterium purity,
Merck) was used as solvent for the (30,40,60-O-tri-
myristoyl-uridine and 30,40,60-O-tri-stearoyl-uridine) and
dimethylsulfoxide (DMSO-d6, 99% deuterium purity, Mer-
ck) employed as solvent for the 30,40,60-O-triacetyl-uridine,
as reported elsewhere.[16,17]
Synthesis of tri-acyl uridine derivatives
30,40,60-O-triacetyl uridine or uridine triacetate
The uridine and nucleophilic catalyst (DMAP) were dis-
solved in a solution of acetic anhydride/DMF/TEA, the
mixture was stirred at room temperature (the detailed
conditions of synthesis are reported elsewhere.[17]). The
product was isolated as a yellow solid (quantitative
yield), melting point: 127–128 °C. 1H NMR (300 MHz;
DMSO-d6), d ppm (multiplicity; integration; J (Hz);
position): 2.04 (d; 6H; H-2″ acetyl linked to O-30 and
O-60 ribose ring), 2.07 (s; 3H; position H-2″ acetyl
linked to O-40 ribose ring), 4.25 (m; 3H; H-60 and H-50
ribose ring), 5.33 (m; 1H; H-40 ribose ring), 5.44 (m;
1H; H-30 ribose ring), 5.72 (d; 1H; J = 8.0; H-5 uracil
ring), 5.87 (d; 1H; J = 5.1; H-20 ribose ring), 7.69 (d;
1H; J = 8.1; H-6 uracil ring). 13C NMR spectroscopy
data and 2D correlations as reported previously.[17] ESI-
MS (positive mode) m/z: Calculated for C15H18N2O9:
370.3114; found, 393.1 [M+Na]+.
Calorimetric analysis
The DSC analysis was performed using a DSC1 Mettler
Toledo Star system equipped with a calorimetry cell and
employing Mettler Star software (11.1 version). The sensi-
tivity was automatically chosen as the maximum available
in the calorimetric system. The reference was prepared with
120 ll of TRIS buffer solution (50 mM, pH = 7.4) in DSC
aluminium pan (160 ll of total volume), hermetically
sealed.
MLV preparation
The phospholipid and tri-acyl ester derivatives of uridine
were separately dissolved in a chloroform/methanol (1 : 1,
v : v) mixture. The DMPC-MLV were prepared in the
absence and presence of uridine or derivatives (Figure 1) at
different molar fractions (0.015, 0.03, 0.045, 0.06, 0.09,
0.12, 0.15 and 0.18) as follows: aliquots of the compound
solutions were added on glass tubes (which contained 7 mg
of phospholipid), in appropriates amount to have the cho-
sen molar fraction. The solvents mixture was removed
under nitrogen flow (in a water bath at 37 °C) to obtain
the lipid films; then, the glass tubes with the lipid films were
subjected to freeze drying to remove residual solvents.
168 ll of TRIS solution (50 mM) at pH = 7.4 were added
30,40,60-O-trimyristoyl uridine and 30,40,60-O-tris-
tearoyl uridine
The synthesis of the tri-acyl ester derivative of uridine with
fatty acids (myristic acid and stearic acid) was performed as
reported elsewhere.[16,17] The fatty acid and coupling agent
(DCC) were dissolved in a DMF/chloroform mix, after the
nucleoside (300 mg) and DMAP (catalyst amount) were
added to reaction mixture. Detailed information of synthe-
sis as reported elsewhere.[17]
30,40,60-O-trimyristoyl uridine was isolated as a white
solid (20% yield), melting point: 63–64 °C. 1H NMR
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Jhon Fernando Berrıo Escobar et al.
Uridine prodrugs/DMPC bilayers interaction
Figure 1 Chemical structure of compounds: (a) uridine; (b) triacetate uridine; (c) trimyristoyl uridine; and (d) tristearoyl uridine.
on each sample that were heated at 37 °C in a water bath
and vortexed 1 min (three times) and left at 37 °C for
1 h.[7,21]
temperature of pure DMPC-MLV. DDH = DH ꢀ DH0,
where DH is the enthalpy variation of MLV prepared in the
presence of the substances and DH0 is the enthalpy varia-
tion of pure DMPC-MLV.
Compounds-MLV interaction studies
Statistical analysis
An aliquot of 120 ll of MLV aqueous dispersion (without
or with the evaluated compounds) was put into an alu-
minium calorimetric pan of 160 ll that was hermetically
sealed and submitted to DSC analysis. The heating scans
were between 5 and 37 °C with heating rate of 2 °C/min.
The cooling scans were between 37 and 5 °C with cooling
rate of 4 °C. The calorimetric scans (heating-cooling) were
carried out at least three times in the temperature range
indicated.[26]
Statistical analysis of data was performed using the
Kruskal–Wallis and the Dunn’s tests. A probability, P, of
less than 0.05 was considered significant.
Results and Discussion
Chemical
The third curve of the heating scans was taken for analy-
sis and calculation of the thermotropic parameters involved
in the phase transitions. Calculation models employed for
the graphics are as follows: DT = Tm ꢀ T0m; where Tm is
the transition temperature of DMPC-MLV prepared in the
The tri-acyl ester derivatives of uridine with aliphatic acid
of short chain were obtained with quantitative yield in brief
time thanks to the reaction for the triacetate of uridine for-
mation carried out in acetic anhydride as solvent and reac-
tive and the use of TEA and DMAP as nucleophilic
catalysts.[17]
presence of the substances and T0 is the transition
m
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Uridine prodrugs/DMPC bilayers interaction
The Steglich method allowed the esterification of all
three hydroxyl groups of the nucleoside, the products were
obtained with yields about 20% for the fatty acids. The
low yields were possible because this method required
the O-acyl-isourea intermediate formation among the car-
boxylic acid and the N,N0-dicyclohexylcarbodiimide (cou-
pling agent), the intermediate showed on the carbonyl
group a reactivity similar to anhydride derivative. More-
over, this compound presented a bulky structure; therefore,
the steric effects were higher and the effective shocks
between intermediate and nucleophile (hydroxyl groups of
the uridine) decreased.[16]
peak of DMPC-MLV. This behaviour suggests that the
compound, due to the polar nature, presents a strong inter-
play with the polar phase (formed by the phosphatidyl-
choline head of DMPC) of phospholipid multilamellar
vesicles, but does not with the lipophilic phase (formed for
the acyl chain of the DMPC). Such an interaction produces
a mobility and change in the acyl chain inclination of the
phospholipid molecules.[21]
The triacetate uridine produces a small decrease in the
enthalpy of the main transition starting from 0.06 molar
fraction (Figure 3b), which indicates that this derivative
presents a weaker interaction with the lipophilic phase of
the phospholipidic bilayers (Figures 2b and 3b). Neverthe-
less, the attractive interactions between the three sub-
stituent groups of the derivative and the myristoyl chains of
phospholipid are weak because the acetyl chain is very short
and the lipophilic character of the tri-acetyl ester of uridine
is weak. Because of this, the compound can penetrate to a
small extent the lipophilic phase and interplay only with a
small portion of acyl chains.[21]
DSC analysis
The aim of this research was to investigate the interac-
tion among the nucleoside-type substances (i.e. uridine
and its derivatives) and the phospholipid bilayers of
MLV and the miscibility between uridine derivatives
and DMPC-MLV. Therefore, MLV of DMPC were pre-
pared in the absence and presence of these compounds
at different concentrations and submitted to calorimet-
ric analysis. The calorimetric curves generated through
the DSC analysis are provided according to the increase
in molar fractions for the compounds evaluated. The
DSC thermogram of the DMPC-MLV typically shows: a
first peak around 14.5 °C corresponding to the pre-
transition (Figure 2), which is related to the transition
from the lamellar gel phase (in this phase the phospho-
lipid chains are mainly distributed in trans configura-
tion) to the rippled gel phase (in this phase the surface
From the thermograms of DMPC-MLV prepared with
the tri-acyl derivatives with fatty acyl chain, it can be easily
observed that these products cause a strong effect on the
thermotropic behaviour of the DMPC-MLV (Figure 2c and
2d). The thermograms related to both substances are char-
acterized by the complete disappearance of the pre-transi-
tion peak starting from lowest molar fraction (0.015)
implicating that these compounds strongly affect the polar
phase of the MLV phospholipids; thereby, causing changes
on the tilt of phospholipid molecules and the configuration
of the acyl chains (Figure 2). Consequently, trimyristoyl
and tristearoyl uridine present a strong interaction with the
phosphatidylcholine head of DMPC-MLV, being possible
only if the nucleoside part, particularly the uracil ring, is
preferably localized or oriented towards the polar phase.[20]
The thermogram of MLV with trimyristoyl uridine sug-
gests a strong effect of this compound on the thermotropic
behaviour of the DMPC-MLV (Figure 2c). The compound
causes a visible shift of the main transition peak toward
lower temperature beginning from the smallest concentra-
tion employed. The effect of the derivative implies that
some moiety of the molecule is incorporated in the lipophi-
lic phase of the bilayers, but this part should be of lipophilic
nature; therefore, the myristoyl chains of the tri-acyl ester
derivative are embedded between the acyl chains of the
phospholipid bilayers. In addition, this substance, as its
concentration increases, produces a gradual broadening of
the main peak and decreasing of the enthalpy associated
with this, starting from the lowest molar fraction used until
the nearly complete disappearance of transition peak at the
highest concentration employed (Figure 3b). This means
that the aliphatic chains of derivative strongly interact with
the acyl chains of the DMPC-MLV; this interaction
of phospholipidic bilayer shows
a periodic rippled
structure) and this behaviour is predominantly associ-
ated with changes in the interaction force on the polar
phase; a second and main peak at around 25 °C (Fig-
ure 2) related to the transition from the rippled gel
phase to a disordered state (called crystal liquid phase)
associated mainly to the lipophilic interaction among
the acyl chains of DMPC-MLV.[28,29] Therefore, the
main peak transition can be employed to have infor-
mation on the nature and strength of interactions in
the system composed of DMPC-MLV and exogenous
compounds.[20]
The DSC thermograms of DMPC-MLV in the absence
and presence of uridine and its uridine triacetate at differ-
ent molar fractions are shown in Figure 2a and 2b. The two
compounds exerted an insignificant effect on the MLV
thermogram. The pre-transition peak remains almost
unchanged. The 30,40,60-O-triacetyl uridine caused only a
small broadening of the pre-transition peak with (starting
from 0.045 molar fraction).
The nucleoside and the tri-acetyl ester derivative exerted
just an imperceptible effect also on the main transition
© 2018 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2018), pp. **–**
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Jhon Fernando Berrıo Escobar et al.
Uridine prodrugs/DMPC bilayers interaction
Figure 2 Differential scanning calorimetry thermograms, in heating mode, of L-a-dimyristoylphosphatidylcholine-multilamellar vesicles prepared
in the presence of increasing molar fractions of uridine (a), uridine triacetate (b), trimyristoyl-uridine (c) and tristearoyl-uridine (d). The X axes
report the temperature (°C).
prevents the cooperativity between the acyl chains of phos-
pholipid molecules in the transition from gel to liquid crys-
talline, thus causing enlargement of the peak. Moreover,
starting from the molar fraction 0.09, a second signal
appears. The temperature of this signal (at lower tempera-
ture) remains almost unchanged but its intensity decreases
in favour of the peak at higher temperature. This can be
due to a not uniform distribution of the derivative inside
the phospholipid bilayers and, consequently, to the pres-
ence of two different phospholipid regions: one reach in
the compound and the other poor in the same.[30,31] These
results are in good agreement with those obtained in a
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Jhon Fernando Berrıo Escobar et al.
Uridine prodrugs/DMPC bilayers interaction
which induces a drastic decrease in the cooperativity pre-
sents between the acyl chain of phospholipid molecules in
the MLV.
The shift of the main transition peak towards higher
temperature shown for the tristearoyl uridine indicates an
unusual thermotropic behaviour of DMPC-MLV when the
exogenous substance interacts with the centre of the phos-
pholipid and seems to stabilize this; therefore, the tempera-
ture of the main transition is higher. This is possible if the
system formed by the lipophilic chains of derivative and
acyl chains of phospholipid are interdigitated forming a
complex structure.[27] The DSC thermograms (Figure 2b)
for the tristearoyl uridine did not show the appearance of a
second peak into the main transition, as observed in the
calorimetry curves related to tri-myristoyl uridine (Fig-
ure 2a). This is possibly due to the fact that tristearoyl uri-
dine uniformly distributed in the multilamellar vesicles
(rich and poor regions of the compound inside the phos-
pholipidic bilayers did not form). Moreover, this derivative
causes a significant decrease in the enthalpy associated with
main transition with the increase in the amount of the
compound employed (Figure 2b), until the complete dis-
appearance of the transition peak at the highest molar frac-
tion employed in this work. The correlation between the
effect exerted by the tested derivatives on the biomembrane
model, and their characteristics clearly indicate an
improved incorporation efficiency of the derivatives with
respect to uridine into the biomembrane model; with tris-
tearoyl uridine and trimyristoyl uridine having the highest
incorporation efficiency, probably due to their stronger
lipophilic character.
Figure 3 (a) Transition temperature variation, as DT/T0
, of
m
L-a-dimyristoylphosphatidylcholine-multilamellar vesicles, as a function
of the molar fraction of compounds evaluated. (b) enthalpy variation,
as DDH/DH0, of L-a-dimyristoylphosphatidylcholine-multilamellar vesi-
cles prepared in the presence of the molar fraction of compounds
evaluated.
The temperature transition and enthalpy variation values
were analysed using the Kruskal–Wallis test. The test con-
firmed the results significance with P < 0.05. The data were
also analysed using the Dunn’s test. Regarding the transi-
tion temperature results, the test indicated as of high signif-
icance the differences between uridine and tristearoyl
uridine, triacetyl uridine and trimyristoyl uridine, trimyris-
toyl uridine and tristearoyl uridine. With regard to the
enthalpy variation values, the Dunn’s test indicated that the
differences between uridine and tristearoyl uridine and
between triacetyl uridine and tristearoyl uridine are highly
significant.
recent study on the interaction between uridine-trimyris-
tate and DPPC and DPPS phospholipid monolayers as
non-tumorogenic and tumorogenic biomembrane cell
models, respectively, prepared using the Langmuir-Blodgett
technique.[23]
The tri-acyl ester derivative of uridine with stearic acid
presented a strong effect on the thermotropic behaviour of
DMPC bilayers. In the thermograms, it is easily observed a
great shift of the main transition peak toward higher tem-
perature and a strong broadening, starting from the small-
est concentration used. The great effect caused by
tristearoyl uridine indicates a strong interaction of the lipo-
philic part of compound with the lipophilic part of DMPC.
Also, the enthalpy associated with the transition from the
gel to the liquid crystalline phase decreased with increasing
tristearoyl uridine molar fractions (Figures 2b and 3b).
These results are due to the strong interaction among the
lipophilic chains (belonging to fatty acid) of the derivative
and the acyl chains of the phospholipids of the bilayers,
Conclusion
The lipophilic character acquired by the tri-acyl ester
derivatives of uridine nucleoside was possible to be
achieved through the conjugation reactions with aliphatic
acid of long chain on the all hydroxyl groups of ribose.
The aliphatic chains of fatty acid linked to the nucleoside
through ester bonds produced an affinity with the lipo-
philic phase of DMPC-MLV used as biomembrane
© 2018 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology, ** (2018), pp. **–**
7

ꢀ
Uridine prodrugs/DMPC bilayers interaction
Jhon Fernando Berrıo Escobar et al.
model. The effect of uridine and tri-acetate uridine on
the thermotropic behaviour of DMPC-MLV was the one
expected for these compounds that indicates their inter-
action with the phospholipid polar head and their loca-
tion within the polar phase of the multilamellar vesicles.
The effect of trimyristoyl uridine and tristearoyl uridine
on DMPC-MLV indicates that a strong interaction of the
compounds and the lipophilic part of DMPC occurs. The
extent of the interaction depends on the length of the
alkyl chain.
biomembrane model, in view of possible future in vivo
studies.
Declarations
Conflict of interests
The authors state that there is no conflict of interests in this
work.
The obtained results highlight the usefulness of the DSC
technique used in this study in helping to understand uri-
dine derivatives insertion and interaction in/with lipid
assemblies such as those of biological membrane; more-
over, to study the performance of different molecules with
Funding
Authors thank financing to the University of Antioquia
(Project CODI CIQF-155 2012–2014, Strategy of Sustain-
ability 2015–2016).
anticancer nucleosides: synthesis and
antitumoral evaluation. Bioorg Med
(Ecyd). Med Chem 2005; 13: 1249–
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