O R I G I N A L P A P E R
Helena FerreiraÆ Marlene Lu´cioÆJose´ L. F. C. Lima Carla Matos ÆSalette Reis
Effects of diclofenac on EPC liposome membrane properties
Received: 27 December 2005 / Revised: 2 March 2005 / Accepted: 8 April 2005 / Published online: 28 June 2005 Springer-Verlag 2005
Abstract In this work the interaction of a non-steroidal anti-inflammatory drug (NSAID), diclofenac, with egg yolk phosphatidylcoline (EPC) liposomes, used as cell- membrane models, was quantified by determination of the partition coefficient. The liposome/aqueous phase partition coefficient was determined by derivative spec- trophotometry, fluorescence quenching, and measure- ment of zeta-potential. Theoretical models based on simple partition of the diclofenac between two different media, were used to fit the experimental data, enabling the determination of Kp. The three techniques used yielded similar results. The effects of the interaction on the membrane’s characteristics were further evaluated, either by studying membrane potential changes or by effects on membrane fluidity. The liposome membrane potential and the size and size-homogeneity of liposomes were measured by light scattering. The effects of dic- lofenac on the internal viscosity or fluidity of the membrane were determined by use of spectroscopic probes—a series of n-(9-anthroyloxy) fatty acids in which the carboxyl terminal group is located at the interfacial region of the membrane and the fluorescent anthracene group is attached at different positions along the fatty acid chain. The location of the diclofenac on the membrane was also evaluated, by fluorescence quenching using the same series of fluorescent probes.
Because the fluorescent anthracene group is attached at different positions along the fatty acid chain, it is pos- sible to label at a graded series of depths in the bilayer.
The interactions between the drug and the probe are a
means of predicting the location of the drug on the membrane.
Keywords Diclofenac ÆLiposomes ÆDerivative spectrophotometryÆ Fluorescence quenchingÆ AnisotropyÆZeta-potential
Introduction
Non-steroidal anti-inflammatory drugs (NSAID) are a group of molecules widely employed in the treatment of inflammatory diseases. In addition to a well-recognized inhibitory effect on cyclooxygenase and lipooxygenase activity [1, 2], NSAID have been reported to affect a variety of other processes, for example cell-membrane function, membrane fluidity [3–6], and above all to in- hibit the generation of reactive oxygen from stimulated neutrophils [7]. There is consensual evidence that lipid affinity of NSAID is of major significance to their toxic and therapeutic action. Indeed, depending on their hy- drolipophilic character, NSAID can be distributed be- tween the membrane and the aqueous phases. This distribution determines their concentration in each phase and thereby controls the extents of their pene- tration into the membrane and/or interactions with phospholipids or other membrane components, for example COX enzymes, which are embedded in the lipid bilayers. Thus, to study the mechanism of action of NSAID and their side effects it is of great importance to investigate the interactions between these drugs and biomembranes.
These interactions can be studied in membrane models, for example liposomes, because these can mimic the chemical and structural anisotropic environment of cell membranes. In this paper, we used unilamellar liposomes (LUV) of egg-yolk phosphatidylcholine (EPC), because the distinct bilayer configuration of the zwitterionic phosphatidylcholine in vesicles seems to mimic the interfacial character and the ionic, H-bond,
H. FerreiraÆM. Lu´cioÆJ. L. F. C. LimaÆS. Reis (&) REQUIMTE/Dep. de Quı´mica–Fı´sica, Faculdade de Farma´cia, Universidade do Porto, Rua Anı´bal Cunha 164,
4050-047 Porto, Portugal E-mail: shreis@ff.up.pt Tel.: +351-2-22078966 Fax: +351-2-22004427 C. Matos
REQUIMTE/Faculdade Cieˆncias da Sau´de,
Universidade Fernando Pessoa, Rua Carlos da Maia 296, 4200-150 Porto, Portugal
DOI 10.1007/s00216-005-3251-z
dipole–dipole and hydrophobic interactions, which may define partitioning in real biomembranes. The combi- nation of partition coefficients and acidity constants to assess possible electrostatic contributions to the inter- action of drugs with liposomes is also a useful approach [8, 9]. Determination of liposome/water partition coef- ficients (Kp) has been achieved by use of different experimental techniques—derivative spectrophotome- try, fluorescence quenching, and measurement of zeta- potential. Fluorescence quenching has also been used to determine the location of the drug. In this study a series of n-(9-anthroyloxy)stearic acids (n-AS, n=2, 6, 9 and 12), which contain the same fluorescent group bonded at different positions of an alkyl chain, was used. Thus, the fluorophores report the environment at a graded series of depths within the host lipid structure, making possible precise mapping of the NSAID according to differences in quenching efficiencies [10].
By use of the zeta-potential technique it was also possible to evaluate the interaction of diclofenac with liposomes by measuring the membrane potential arising from drug partitioning. Indeed, zeta-potential measure- ments are important in the determination of the effect of the drugs on membrane surface potential, enabling characterization of the electrostatic membrane proper- ties induced or altered by drug binding. This therefore gives an indication of the extent of interactions that exist between drugs and the lipid bilayer surface.
Finally, perturbation of the bilayer structure at dif- ferent depths induced by the NSAID, which can give useful information about the physical state of bio- chemical membranes, was measured by steady-state anisotropy [11,12]. In these studies the same set ofn-AS probes were used, because they enable study of the flu- idity gradient in model membranes [13–15]. Although steady-state fluorescence anisotropy has proved useful for monitoring structural changes in biomembranes, it was necessary to consider appropriate corrections to experimental data to enable clear interpretation of the results.
Materials and methods
Reagents and equipment
The anti-inflammatory drug diclofenac, EPC, and (±)- 12-(9-anthroyloxy)stearic acid (12-AS) were from Sigma;
the other probes, (±)-2-(9-anthroyloxy)stearic acid (2-AS), (±)-6-(9-anthroyloxy)stearic acid (6-AS), and (±)-9-(9-anthroyloxy)stearic acid (9-AS) were from Molecular Probes; all were used as supplied. All other chemicals were from Merck (pro analysi). Solutions were prepared in double-deionized water (conductivity less than 0.1 lS cm1), and for all solutions studied the ionic strength was adjusted to 0.1 mol L1with NaCl.
Absorption spectra were recorded at 25.0±0.1C with a Perkin–Elmer Lambda 45 UV/Vis spectropho- tometer in the range 220–500 nm at 1 nm intervals (a
Hitachi U-2000 dual-beam spectrophotometer, a 220–
400 nm range at 2 nm intervals was used for determi- nation of acidity constants). In all cases, quartz cells were used and the temperature was kept constant by circulating thermostatted water in the cell holder.
Fluorescence studies were performed with a Perkin–
Elmer LS 50B steady-state fluorescence spectrometer equipped with a constant-temperature cell holder. All data were recorded at 25.0±0.1C using 1-cm cuvettes.
The excitation wavelength was set to 384 nm; the emis- sion wavelength was set to 446 nm for 12-AS and for 9-AS and to 451 nm for 6-AS and 2-AS. Fluorescence intensity data were corrected for absorbance of the quencher (diclofenac) at the excitation wavelength [16].
The zeta-potential (f-potential) values and the size distribution of extruded EPC liposomes, with and without incorporated drug, were determined at pH 7.4 (Hepes buffer), at 25.0±0.1C, by quasi-elastic light scattering analysis using a ZET 5104 cell in a Malvern ZetaSizer 5000, with a 90scattering angle.
Determination of acidity constant by spectrophotometry The acidity constant of diclofenac was obtained from UV/Vis data, at different pH, in aqueous solution of ionic strength adjusted to 0.1 mol L1 by addition of NaCl. The pH was measured by means of an electrode system comprising an Orion 900029/4 AgCl/Ag refer- ence electrode and a Russell SWL glass electrode. Sys- tem calibration was performed by the Gran method [17], in terms of hydrogen ion concentration, by titrating solutions of strong acid (103mol L1HCl) with strong base (0.02 mol L1NaOH). The acidity constants were obtained by titration of 25 mL of an alkaline aqueous solution (with NaOH 50%) of the drug (approximately 40lmol L1) with HCl (titrisol 103mol L1), added with an automatic piston burette, controlled by a per- sonal computer, under a nitrogen stream. The absorp- tion spectra were recorded with the system described in
‘‘Reagents and equipment’’. A peristaltic pump enabled circulation of the solution from the reaction pot, through a flow-cell of the spectrophotometer and back to the reaction pot.
Calculations were performed with data obtained from at least two independent experiments using the program pHab [18].
Liposome preparation and drug incorporation
Liposomes were prepared by the thin film hydration method [19]. A known amount of EPC was dissolved in chloroform–methanol (9:1), the organic solvent was evaporated under a stream of nitrogen, and the lipid film formed was then left under vacuum at least 3 h to re- move traces of the organic solvents. The resulting dried lipid film was dispersed in buffered solutions of different pH: hydrochloric acid (34.6 mmol L1)–glycine
(50 mmol L1) (pH 3.0); Hepes (pH 7.4; 10 mmol L1);
sodium hydroxide (43.2 mmol L1)–boric acid (50 mmol L1) (pH 10.3). In all experiments I=0.1 mol L1, and for pH 3.0 and 10.3 the final pH was adjusted by addition of strong acid (HCl) or base (NaOH). The mixture was vortex mixed above the phase-transition temperature (room temperature), to yield multilamellar vesicles, which were then extruded ten times through polycarbonate filters of 100 nm pore size (Nucleopore) to form large unilamellar vesicles (LUV) [19]. The EPC concentration in vesicle suspen- sions was determined by phosphate analysis using the phosphomolybdate method [20].
In the fluorescence measurements, the fluorescence probes were dissolved in ethanol and added to a sus- pension of pre-formed LUV, with gentle mixing. The lipid-to-probe ratio was always greater than 100:1 to prevent changes in the structure of the liposome mem- branes. To ensure complete incorporation of the probe into the lipid bilayer the suspensions were left to stand in the dark for 30 min [21].
After liposome preparation and/or labelling the drug samples were prepared by mixing a known volume of drug solution and a suitable amount of vesicle suspension in buffer; the corresponding reference solutions were pre- pared identically, but without drug. All suspensions were then vortex mixed and incubated at room temperature.
Determination of partition coefficients by derivative spectrophotometry
The partition coefficient (Kp) of diclofenac was deter- mined in LUV suspensions at pH 3.0, 7.4 and 10.3. In the derivative spectrophotometric studies a series of buffered suspensions containing a fixed concentration of drug (40lmol L1) and increasing concentrations of EPC (in the range 70–1200lmol L1) were prepared.
The corresponding reference solutions were prepared identically but without drug. All suspensions were then vortex mixed and incubated at room temperature in the dark for 30 min; after equilibration the absorption spectra were recorded.
Determination of partition coefficients and drug location, and membrane fluidity studies
by fluorescence measurements
Partition coefficient and drug location were determined by fluorescence quenching, and membrane fluidity was estimated by fluorescence anisotropy. For determination of Kp, liposomes were prepared with the 2-AS probe included. In drug location and membrane fluidity stud- ies, unlike the previous determination all n-AS fluores- cence probes in LUV suspensions at pH 7.4 were used.
For determination of Kp, the EPC concentration of the liposomes ranged between approximately 80 and 900lmol L1, and for each lipid concentration a series
of crescent drug concentrations was prepared. In the other studies all the suspensions contained the same amount of lipid (approximately 500lmol L1). Hepes (pH 7.4, 10 mmol L1, I=0.1 mol L1)-buffered solu- tions of diclofenac were added to the liposomes prepared with the n-AS probe as previously described. Drug concentrations were in the range 0–500lmol L1.
The resulting suspensions were vortex mixed and incubated in the dark for 1 h.
Determination of zeta-potential and size
Zeta-potential (f-potential) values and size distribution of EPC liposomes, with and without incorporated drug, were determined at pH 7.4 (Hepes buffer), at 25.0C.
Lipid concentration was kept constant at approximately 400lmol L1. Diclofenac concentrations ranged be- tween 0 and 500lmol L1. The values for viscosity and refractive index were taken as 0.890 cP and 1.330, respectively [22]. All suspensions were then vortex mixed and incubated in the dark for 30 min; after equilibration the zeta-potential and size measurements were recorded.
The mean particle size of the vesicles was found to be 133±5 nm (average and standard deviation from mea- surements of six independently prepared suspensions).
Results and discussion
Determination of acidity constant by spectrophotometry The pKaobtained for diclofenac was 3.97±0.04, a value similar to that previously published (3.99 at 25C) [8], which is associated with the carboxyl group. Application of the Henderson–Hasselbalch equation reveals that at the physiological pH (7.4) diclofenac exists 99.9% in the anionic form, whereas at pH 3.0 the uncharged form predominates.
Determination of partition coefficients by derivative spectrophotometry
The derivative UV spectra of diclofenac show a decrease in absorption intensities in the presence of increasing amounts of EPC. Furthermore, the spectra contain isosbestic points and a bathochromic shift in kmax (Fig.1) is evident, an observation that suggests that the environment of the molecules has become less polar, implying interaction between the drug and the non-polar hydrocarbon bilayers of the liposomes [23,24].
The partition coefficient (Kp) of a compound between a liposome suspension and the aqueous solution is re- lated to the absorbance intensities by the expression [25]:
AbsT¼AbswþðAbsmAbswÞKp½ VL /
1þKp½ VL /
ð1Þ
where AbsT, Absm, and Absw are the total, lipid and aqueous absorbances of the drug, respectively,Kpis the partition coefficient, [L] the lipid concentration andV/is the lipid molar volume. For EPC, V/=0.688 L mol1 and the mean molecular weight is 700 g mol1[26].
A formally identical expression can be used for derivative spectroscopy, with Abs replaced by D=(dnAbs)/(dkn). This equation was fitted to the experimental data (DT versus [L]), using a non-linear least-squares regression method (Fig.2).
Partition coefficients were determined from the sec- ond and third derivative spectra (calculated from the recorded spectra after blank subtraction), at wave-
lengths where the scattering is eliminated. The values of Kp obtained and their experimental error are listed in Table1.
The much higher value of Kp at pH 3.0 (26,000±3000) reflects the hydrophobicity of the neutral form of diclofenac that exists predominantly in this form in acidic media. In contrast, at pH 7.4 and 10.3, the drug is almost completely ionized and the Kp values are identical and much smaller, a consequence of its charge.
Determination of partition coefficients by fluorescence quenching
Fluorescence quenching can be described by the classical Stern–Volmer equation [10, 27–31]. However, when the quencher is distributed between the membrane and the aqueous phase and only the total diclofenac concentra- tion, [Q]T, is known, the Stern–Volmer equation can be written as [10]:
Table 1 Partition coefficients (dimensionless) for diclofenac in EPC unilamellar liposomes (LUV), by derivative spectrophotometry, fluorescence quenching, and zeta-potentiala
Method Kp
Derivative spectrophotometry 26,000±3,000 (pH 3.0) 1,200±100 (pH 7.4) 1,100±50 (pH 10.3) Fluorescence quenching 1,170±200 (pH 7.4)
Zeta-potential 1,070±320 (pH 7.4)
aThe reported values are the mean of at least three independent measurements; the error that affects each value is the standard deviation
Fig. 2 Second-derivative spectrophotometric data at k=322 nm for diclofenac in egg yolk phosphatidylcholine (EPC) at different concentrations (0, 82, 160, 262, 342, 423, 547, 622, 706, 809, 889, 961 and 1076lmol L1). Thecurverepresents the best fit to Eq. 1 Fig. 1 Second derivative
spectra of diclofenac in egg yolk phosphatidylcholine (EPC) at different concentrations:10, 282,3160,4262,5342,6423, 7547,8622,9706,10809, 11889,12961,13
1076lmol L1
I0
I ¼1þKsvapp½ QT ð2Þ
in which I0 and I are, respectively, the corrected fluo- rescence intensity of the fluorophores (n-AS probe) in the absence and presence of the drug, and Ksvapp is the apparent Stern–Volmer constant. The values of this constant were determined from the slope of the Stern–
Volmer plots.
Besides depending on the quencher efficiency, the Ksvapp values also depend on its partition coefficient (Kp) between the aqueous and the lipid phases. This depen- dence can be described by the equation [10]:
Ksvapp¼Ksv
Kp
KpVmþ1 ð3Þ
By fitting Eq. 3 to sets of values Ksvapp;Vm
using a non- linear regression method (Fig.3) it is possible to deter- mine the quencher partition coefficient and the Stern–
Volmer constant (KSV). The results obtained with this methodology (Table1) are in agreement with the results provided by the other experimental techniques.
Zeta-potential determinations
From the experimentally determined zeta-potential val- ues (f), it is possible to calculate the surface potential (W0), which in turn enables calculation of the surface charge density on the membrane (r), using the Gouy–
Chapman theory [32, 33]. The complete mathematical formalism was described in a previous paper [22].
Surface charge density on the membrane, r, can be expressed as the number of charged molecules per unit area, denoted hereafter as r*, by the expression r*=rN//F, in whichNis the Avogadro number andFis
the Faraday constant [22]. Ther* value is related to the molar concentration of charged drug in the membrane
Am
;by [22]:
A -
m
¼ r½ aL L
1raA
ð4Þ whereaLis the molecular surface area of the lipid andaA
is the area of the drug.
Once the value of A-
m
is known, calculation of the concentration of free drug in the aqueous phase, A -
w
; is straightforward using a mass balance
AT ¼ A-
m
þAw
; assuming the total amount of drug added to the system, [AT] is known and the value of the partition coefficient,Kp, can be obtained using Eq. 5:
Kp¼ATm ATw
ð5Þ The value ofaLis described in the literature as 60 A˚2 [34], but the molecular surface area of diclofenac, aA
could not be found, and we have assumed a value of 60 A˚2, because this quantity does not affect significantly the results obtained—the value obtained for Kp was 1069±322 with aA=60 A˚2 and 1063±325 with aA=20 A˚2.
Results for zeta-potential (f) measurements, obtained at increasing diclofenac concentrations, in Hepes buffer (pH 7.4, 10 mmol L1), show an increase from 1.0 mV, for drug-free liposomes to 17.5 mV for 500lmol L1 diclofenac. This observed membrane charging is a direct consequence of negative drug par- titioning in the membrane, because diclofenac is almost entirely (99.9%) ionized at this pH. At pH 3.0, increasing concentrations of diclofenac had no effect on membrane surface potential, because at this pH dic- lofenac’s uncharged form predominates.
The fvalues obtained were corrected by subtracting the zeta-potential of EPC in the absence of drug, and used to calculater, by the Gouy–Chapman theory [32, 33], and, as described previously Am
and A-
w
AT ¼Am
þAw
: As the lipid concentration is fixed and known,Kpcan be calculated from Eq. 5.
However, the partition of negatively charged species, A, will lead to charging of the membrane that will repel ions of the same charge in the aqueous solution so that, at equilibrium, the concentration of the charged form at the interface Ai
will be lower than its bulk concen- tration, A -
w
:Because only drug in the vicinity of the membrane can establish partition equilibrium, the Kp
value is calculated using Ai
which can be obtained using the Boltzmann equation [22,35].
Results show that the values obtained without this correction are lower than the corrected values, and that the differences are larger as the drug concentration in- creases. For a drug concentration below 50lmol L1, however, the two sets of results are similar within experimental error, suggesting that the influence of electrostatic effects on partition coefficients can be
Fig. 3 Apparent Stern–Volmer constants KSVapp
of 2-AS for diclofenac in egg yolk phosphatidylcholine (EPC) unilamellar liposomes (LUV) obtained for different lipid concentrations ([L]).
Thecurverepresents the best fit to Eq. 3
neglected for low drug concentrations. Results presented in Table 1are averages of values obtained for concen- trations below 50 lmol L1, which are of the order of magnitude of the drug concentrations used in the other two presented techniques.
Finally, the knowledge of the maximum number of charged molecules per unit area, rmax; enables calcula- tion of maximum molar ratio lipid/drug n; which is independent of lipid concentration, as [22]:
rmax¼ 1 aAþn aL
ð Þ ð6Þ
The values of rmax can be calculated by fitting a plot of r versus [AT] [36]:
h¼ Kb AT
1þKb½AT ð7Þ
in which h, the degree of saturation, is given by h¼ r=rmax:
The values ofrmaxwere determined by fitting Eq. 7 to the plot ofr versus [AT], as can be observed in Fig.4.
The value of rmax obtained was 1.52±0.14·103 molecules/A˚2(average and standard deviations of three independent experiments). As stated, the value of r reflects only the partition of the negative form of the drug. Calculation of n by use of Eq. 6 requires the knowledge of the lipid and drug molecular surface, aL
andaA, and once more it is focussed that the values are not affected by the value of aA:n =10.1±1.0 with aA=60 A˚2andn=10.8±1.0 withaA=20 A˚2.
Drug location
The fluorescence intensity ofn-AS probes decreases with diclofenac concentration, and Fig.5shows an example of this behaviour for the 12-AS probe in EPC LUV.
By comparing the apparent Stern–Volmer constants for the quenching of the n-AS probes, an indication of how deeply the quencher is buried within the bilayer structure can be obtained [29].
The use of this set of probes to determine drug location in the bilayer is based on the assumption that all probes have the same intrinsic quenching efficiency [29]. This is true in homogeneous solvents, but in phospholipid bilayers the presence of fluidity and polarity gradients through the plane of the membrane is reflected in different s0values found for the probes for different environments [14, 29,37–41]. Consequently, it is advisable to calculate the bimolecular constant,Kq, in which the effect of the microenvironment differences surrounding the probes is eliminated. From the knowl- edge of the determined Kp values and the fluorescence lifetime of each probe at pH 7.4 [42], it is possible to determineKq. By observation of these values (Table2), it was possible to conclude that all n-AS probes were quenched by diclofenac and the relative quenching effi- ciencies follow the order 2-AS12-AS>9-AS6-AS.
This suggests that the anti-inflammatory drug is lo- cated near the phospholipid headgroup, probably by electrostatic adsorption with the zwitterionic head of phosphatidylcholine (as can be seen by the greater quenching efficiency observed with the 2-AS probe). On the other hand, the chemical structure of diclofenac provides an explanation of the high quenching efficiency demonstrated for the 12-AS probe—a strong additional
Fig. 4 Dependence of number of charged molecules per unit area, r
ð Þ, on the concentration of diclofenac at pH 7.4, in the presence of 400lmol L1egg yolk phosphatidylcholine (EPC). The curve represents the best fit to Eq. 7
Fig. 5 Fluorescence quenching of 12-AS probe in egg yolk phosphatidylcholine (EPC) unilamellar liposomes (LUV) (500lmol L1, pH 7.4) by diclofenac
quenching group (chlorine atoms) possibly lies near position 12 of the phospholipid acyl chain.
Membrane fluidity studies
To evaluate possible modulation of membrane fluidity by diclofenac, the steady-state fluorescence anisotropy was measured using fluorescent probes [10]. The method described by Lakowicz [10] was used to measure the steady-state anisotropies, rss, by use of the equation:
rss¼ IvvGIvh Ivvþ2GIvh
ð8Þ in whichGis the instrumental correction factor, andIvv
and Ivh are the emission intensities polarized vertically and horizontally to the direction of the polarized light.
However, changes in anisotropy with increasing concentration of drug can occur as a result of changes either in membrane fluidity or in the excited-state life- time of the fluorophore, s ¢. This latter effect can be eliminated by the use of corrected anisotropy values (r¢) [43]:
r0¼hþs0
hþs0rss ð9Þ
wheres0ands¢are the corrected fluorescence lifetime of the fluorophore in the absence and presence of the drug, respectively, andhis the rotational correlation time.
The values of s¢can be easily calculated by using the following equation [10]:
I0
I ¼s0
s0 ð10Þ
where I0 and I are, respectively, the corrected fluores- cence intensity of the n-AS probes, in the absence and presence of the drug.
Considering, in Eq. 9,rssas the value measured in the absence of quencher and the published values ofhands0 for then-AS probes used [42], it is possible to obtain the values of the corrected anisotropy,r¢. Theser¢values are the variation of anisotropy that should be obtained be- cause of the fluorescence lifetime changes and are then compared with the experimental rss values. When the difference between rss and r¢ increases, it is concluded that a decrease in membrane fluidity occurs [43].
By plotting the values ofrssand the calculated values of r¢ as a function of drug concentration it becomes evident that diclofenac reduces membrane fluidity for
the 2-AS probe, and for the other probes this pertur- bation is not evident (Fig.6).
Only the drug partitioned in the membrane affects the anisotropy [44], and can be obtained from [10]:
Qm
½ ¼ Kp½QT
Kpamþð1amÞ ð11Þ where am is the volume fraction of membrane phase (am=Vm/VT; Vm and VT represent the volumes of the membrane and water phases, respectively).
The effects of diclofenac on membrane fluidity par- allel those of drug location; diclofenac’s carboxyl groups increase the rigidity of the head groups of the phos- pholipids, and this is in agreement with the drug’s preferential location at the surface of the membrane.
This behaviour can be explained by the fact that in the region where the drug is located there would be less disordering, because of an increase in packing density, than in the other regions of the bilayer. The chloro- substituted benzene ring is probably probing the inner region of the bilayer, but does not significantly affect membrane fluidity. The same effect is observed for the high-order plateau region (closer to the 6-AS and 9-AS probes).
Conclusion
In this work we were able not only to quantify the extent of diclofenac–liposome interactions but also able to show that they are controlled by the chemical nature of the pharmaceutical compound. Indeed, depending of their acid–base properties and lipophilicity, the interac- tion can increase or decrease. Therefore, characteriza- tion of the acidity constants is necessary for thorough understanding of the pharmacokinetic and pharmaco- dynamic properties of the drug. As for other compounds [22], small concentrations of diclofenac occurring in the charged form have negligible electrostatic effects on partition coefficients but for higher concentrations these effects increase dramatically, and play a crucial role in the partition of charged forms of diclofenac, and thus partition coefficients must be calculated using surface concentrations. However, when the partition coefficients calculated from zeta-potential (f) measurements are compared with those obtained from derivative spec- troscopy or from fluorescence-quenching determina- tions, they are found to agree within experimental error, at least for the small concentrations used in the latter
Table 2 Values of apparent Stern–Volmer constants KSVapp
and bimolecular rate constants (Kq) obtained for diclofenac in unilamellar liposomes (LUV)a
2-AS 6-AS 9-AS 12-AS
KSVappM - 1
700±20 500±70 610±70 1110±20
Kq108M - 1 s - 1
1.42±0.25 0.93±0.08 0.98±0.06 1.36±0.15
aThe reported values are the mean of three independent measurements; the error is the standard deviation
methods. The data obtained for the location of diclofe- nac are also consistent with zeta-potential studies, given that this drug creates an appreciable decrease of the zeta-potential, an indication that this compound is preferentially located near the phospholipid headgroups.
Another effect of the interaction of diclofenac with EPC liposomes on membrane properties was measured by studying changes in membrane fluidity. These results are also in agreement with previous studies.
Evaluation of the change in the characteristics of different membranes is fundamental. Both electrostatic and fluidity properties can affect the conformation and activity of the membrane, and of membrane-bound en- zymes and several cell processes; this, in turn, can be directly related to inflammatory actions of diclofenac.
Therefore, we can conclude that study of the mech- anism of action of anti-inflammatory drugs, and their side effects, may fall in part in the domain of
membranology, particularly investigation of the nature of the interaction between NSAID and phospholipids.
Finally, it should be remarked that one of the most important achievements of this work was to apply a methodological framework based on common analytical techniques. Hence, it is possible to obtain a large amount of information by using classical analytical tools.
Acknowledgements The authors would like to thank FCT and FEDER for financial support through the contract POCTI/FCB/
47186/2002. Two of us, H.F. and M.L., thank FCT for fellowships BD 6829/01 and BD 21667/99, respectively.
References
1. Kitchen EA, Dawson W, Rainsford KD, Cawston T (eds) (1985) Anti-inflammatory and anti-rheumatic drugs. CRC Press, Boca Raton
2. Vane JR (1971) Nature 231:232–235
3. Beccerica E, Ferretti G, Curatola G, Cervini C (1990) Phar- macol Res 22:277–285
4. Beccerica E, Piergiacomi G, Curatola G, Ferretti G (1989) Pharmacology 38:16–22
Fig. 6 Values of corrected anisotropy, r¢ (grey filled squares) and steady-state anisotropy, rss (black filled squares) for each n-AS probe in egg yolk phosphatidylcholine (EPC) unilamellar lipo- somes (LUV) (500lmol L1, pH 7.4) caused by increasing effective concentrations of diclofenac
5. Knazek RA, Liu SC, Dave JR, Christy RJ, Keller JA (1981) Prostaglandin Med 6:403–411
6. Simonetti O, Ferretti G, Offidani AM, Gervasi P, Curatola G, Bossi G (1996) Arch Dermatol Res 288:51–54
7. Bomalaski JS, Hirata F, Clarke MA (1986) Biochem Biophys Res Commun 139:115–121
8. Balon K, Riebesehl BU, Muller BW (1999) J Pharm Sci 88:802–
806
9. Taka´cs-Nova´k K, Avdeef A, Box KJ, Poda´nyi B, Sza´sz G (1994) J Pharm Biomed 12:1369–1377
10. Lacowicz JR (ed) (1999) Principles of fluorescence spectros- copy. Kluwer Academic/Plenum Press, New York
11. Fendler JH (1980) J Phys Chem 84:1485–1491
12. Miller MA, Sagnella GA, Markandu ND, MacGregor GA (2001) Clin Sci 100:653–658
13. Johns SR, Willing RI, Thulborn KR, Sawyer WH (1979) Chem Phys Lipids 24:11–16
14. Thulborn KR, Sawyer WH (1978) Biochim Biophys Acta 511:125–140
15. Tilley LM, Thulborn KR, Sawyer WH (1979) J Biol Chem 254:2592–2594
16. Coutinho A, Prieto M (1993) J Chem Educ 70:425–428 17. Gran G (1952) Analyst 77:661–671
18. Gans P, Sabatini A, Vacca A (1999) Anal Chim 89:45–49 19. Lasic DD (ed) (1993) Liposomes—from physics to applica-
tions. Elsevier, New York
20. McClare CWF (1971) Anal Biochem 39:527–530
21. New RRC (ed) (1990) Liposomes—a practical approach. Ox- ford University Press, New York
22. Matos C, Castro B, Gameiro P, Lima JLFC, Reis S (2004) Langmuir 20:369–377
23. Kitamura K, Imayoshi N, Goto T, Shiro H, Mano T, Nakai Y (1995) Anal Chim Acta 304:101–106
24. Welti R, Mullikin LJ, Yoshimura T, Helmkamp GM Jr (1984) Biochemistry 23:6086–6091
25. Ferreira H, Lu´cio M, Castro B, Gameiro P, Lima JLFC, Reis S (2003) Anal Bioanal Chem 377:293–298
26. White SH, Jacobs RE, King GI (1987) Biophys J 52:663 27. Blatt E, Ghiggino KP, Sawyer WH (1981) J Chem Soc
177:2551–2558
28. Blatt E, Sawyer WH (1985) Biochim Biophys Acta 822:43 29. Haigh EA, Thulborn KR, Sawyer WH (1979) Biochemistry
18:3525–3532
30. Putilina T, Sittenfeld D, Chader GJ, Wiggert B (1993) Bio- chemistry 32:3797–3803
31. Vermeir M, Boens N (1992) Biochim Biophys Acta 1104:63–72 32. Eisenberg M, Gresalfi T, Riccio T, MacLaughlin S (1979)
Biochemistry 18:5213–5223
33. Winiski AP, Eisenberg M, Langner M, MacLaughlin S (1988) Biochemistry 27:386–392
34. Rooney EK, East JM, Jones OT, McWhieter J, Simmonds AC, Lee AG (1983) Biochim Biophys Acta 728:159–170
35. MacLaughlin S, Harary H (1976) Biochemistry 15:1941–1948 36. Connors KA (ed) (1987) Binding constants. The measurement
of molecular complex stability. Wiley, New York
37. Abrams FS, London E (1993) Biochemistry 32:10826–10831 38. Castro B, Gameiro P, Lima JLFC, Matos C, Reis S (2001)
Colloid Surf A 190:205–212
39. Gazzara JA, Phillips MC, Lund-Katz S, Palgunachari MN, Segrest JP, Anantharamaiah GM, Rodrigueza WV, Snow JW (1997) J Lipid Res 38:2147–2154
40. Thulborn KR, Tilley LM, Sawyer WH, Treloar FE (1979) Biochim Biophys Acta 558:166–178
41. Villalaı´n J, Prieto M (1991) Chem Phys Lipids 59:9–16 42. Vincent M, de Foresta B, Gallay J, Alfsen A (1982) Biochem-
istry 21:708–716
43. Lu´cio M, Ferreira H, Lima JLFC, Matos C, Castro B, Reis S (2004) Phys Chem Chem Phys 6:1493–1498
44. Giuliano F, Ferraz JGP, Pereira R, de Nucci G, Warner TD (2001) Eur J Pharmacol 426:95–103
