Fluoroquinolone distribution in a phospholipid environment studied by spectrofluorimetry

Analytica Chimica Acta, 290 (1994) 58-64 Elsevier Science B.V., Amsterdam

58

Fluoroquinolone distribution in a phospholipid environment studied by spectrofluorimetry M.T. Montero and J . Hernandez-Borrell Unitat de Fisicoquimica, Facultat de Farmacia, Universitat de Barcelona, 08028 Barcelona (Spain)

K. Nag and K .M.W. Keough Department of Biochemistry, Memorial University of Newfoundland, St . John's, Newfoundland, AIB 3X9 (Canada)

(Received 4th May 1993)

Abstract The intrinsic fluorescence of fluoroquinolones was exploited first to discriminate between fluorophore populations after encapsulation in liposomes, and second to observe by epifluorescence their distribution in phospholipid monolayers . The affinity of fluoroquinolones for a hydrophobic environment and especially for phospholipids could help in interpreting the mechanism of drug uptake in bacteria . Additionally, this will lead to improved encapsulation efficiencies of these drugs in liposomes . Keywords: Fluorimetry; Fluoroquinolones ; Liposomes ; Pharmaceutical ; Phospholipids

The quinolones are a group of antibacterial agents derived by systematic modification of 1,4dihydro-4-oxopyridine-3-carboxylic acid . Among them, fluoroquinolones having cyclopropyl, piperazinyl and fluorine substituents at the N-1, C-7 and C-6 positions are the most clinically useful [1]. There is renewed interest in the development of new methods for the determination of these molecules. Although studies on the physicochemistry properties of fluoroquinolones have recently been published [2-5], little information is available on their spectroscopic characteristics [6] . Liposome-encapsulated fluoroquinolone has recently been used against the Mycobacterium avium-M. intracellulare complex [7]. Although UV-visible spectrophotometry is frequently used Correspondence to: M.T. Montero, Unitat de Fisicoquimica, Facultat de Farmacia, Universitat de Barcelona, 08028 Barcelona (Spain) .

to measure fluoroquinolones, spectrofluorimetry has hardly been used for this purpose . Therefore, taking into account the very low concentrations used in microbiological studies on liposomes, the spectrofluorimetric and UV-visible spectrophotometric properties of fluoroquinolones were compared as a basis for developing useful methods for determining membrane- or liposomebound drugs . An object of our research is to encapsulate fluoroquinolones in liposomes with maximum efficiency [8]. Fluoroquinolones are zwitterions with pKe values of ca . 6 and 8 for the amino groups, which are positively charged at acidic pH and render the drugs partially soluble in aqueous media at lower pH [3]. On the other hand, fluoroquinolones are partially hydrophobic, as revealed by the partition coefficients in an n-octanolbuffer system [5]. However, the incorporation of the drugs into a phospholipid environment has

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M. T. Montero et at /Anal Chim. Acta 290 (1994) 58-64

not been well studied . Among the mechanisms of interaction between fluoroquinolones and bacterial cytoplasmic membranes, we are concerned with the binding to phospholipids [9] and uptake by simple diffusion [10]. Therefore, in the second part of this work the localization of the drugs in bilayers was studied by quenching of fluorescence and observing by epifluorescence the distribution of the fluoroquinolone in a phospholipid monolayer spread at an air/water interface . EXPERIMENTAL

L-a-Dipalmitoylphosphatidylcholine (DPPC), specified as 99% pure, was obtained from Sigma (St. Louis, MO) . It was assessed as > 99% pure by thin-layer chromatography [developing solvent CHC1 3-CH3 OH-H 2O-HCO2 H (80 + 25 + 3 + 1), H 2SO4 spray and charring] . The quinolone ITV 8912 ®, 1-cyclopropyl-1,4-dihydro-4-oxo-6fluoro-7-(3-methylpiperacinyl)-3-quinolincarboxylic acid, was synthesized by ITEVE Labs . (Reus, Spain). Buffer A was 1 .74 mM boric acid-10 mM sodium tetraborate (pH 8.9), made 227 mOsm kg -1 (mOsm = milliosmolarity) with NaCl, buffer B was 3 .20 mM KH 2PO4 17 .39 mM NaH 2PO4 H 20 (pH 7 .44), made 261 mOsm kg -1 with NaCl, and buffer C was 0 .34 mM acetic acid-0.84 M sodium acetate (pH 4.70), 1341 mOsm kg -1. Deionized water was distilled from sodium permanganate in an all-glass apparatus and further purified by reverse osmosis through a Milli-Q system (Millipore) . All other chemicals were of analytical-reagent grade or better and all organic solvents were redistilled . Sephadex G-50 gel for chromatography was obtained from Pharmacia (Uppsala, Sweden). Vesicle preparation and determination of encapsulation efficiency

The interior of a 10-ml conical tube was coated with a thin lipid film by evaporation to dryness of a chloroform solution containing 40 µmol of DPPC under a stream of nitrogen . After evacuation of the dried material for > 2 h, multilamellar vesicles (MLVs) were obtained by hydration of the lipid film by vortex mixing in 11 .58 mM quinolone solution in acetate buffer (pH 4.7).

Between vortex mixing periods, heating above the lipid gel to the liquid-crystal transition temperature, Tm, was used to obtain a milky, stable suspension. Subsequently, vesicles were extruded ten times through 100-nm polycarbonate membranes (Nuclepore) following a method described elsewhere [11] . The quinolone content associated with liposomes was determined after the separation of free quinolone from liposome-entrapped quinolone by gel filtration on Sephadex G-50 . After chromatographic separation, an aliquot of the effluent was assayed by liquid chromatography (LC) [8]. Lipid concentrations were determined as described elsewhere [12]. Fluorescence quenching studies

The intrinsic fluorescence of the quinolone was doubly exploited . First, when quantification was needed the quinolone was excited at 278 nm with a slit width of 0 .5 mm and the fluorescence was monitored at 448 run at constant temperature. Second, fluorescence quenching methods were applied to localize the quinolone in bilayers . All fluorescence measurements were carried out using a Hitachi F2000 spectrofluorimeter . Liposomes encapsulating quinolone were incubated at 25 and 50°C (below and above the phase transition temperature of DPPC, Tm = 41°C) in the presence of iodide as a quenching agent . The suspension was placed in a quartz cuvette in the jacketed cuvette holder of the fluorimeter, which was mantained at the desired temperature. Iodide can quench the fluorescence of the fluoroquinolone molecule only when the drug is in hydrophilic regions. To distinguish between two populations of fluorophores a treatment based on a modification of the Stern-Volmer equation [13] was used: FO/OF= 1/fa Ksv [Q] + 1/fa ( 1) where fa is the fraction of the initial fluorescence that is accesible to the quencher Q, AF is the difference between the fluorescence intensity in the absence (F0) and presence (F) of quencher and Kg, is the Stern-Volmer constant. Linear regression equations were determined with the Statworks Package on a Macintosh SE/30 personal computer.

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M. T. Montero et al. /Anal Chim. Acta 290 (1994) 58-64

Epifluorescence monolayer studies

58 x 10-6 M . The excitation and emission spec-

Epifluorescence monolayer studies were car-

tra of the fluoroquinolone were characterized by

ried out using the technique described elsewhere

bands with maxima at 278 and 448 nm, respec-

[14] . Briefly, the lipid and fluoroquinolone were

tively. Whereas the excitation maximum of the

mixed in chloroform-methanol solution with 1

fluoroquinolones was a function of the concentra-

mol% of a fluorescent lipid probe, rhodamine-PE .

tion, the emission maximum remained un-

The material was spread in a monolayer on water

changed . Above 80 AM, further increases in fluo-

in an epifluorescence surface balance [14] and the

roquinolone concentration did not result in any

distribution of lipid into the expanded and con-

detectable change in the excitation or emission

densed phases was monitored by measuring the

wavelengths . These fluorescence findings could

fluorescence of rhodamine-PE . The distribution

be interpreted, as in other cases [15], by the

of the drug could be monitored through its intrin-

formation of fluoroquinolone aggregates at higher

sic fluorescence by appropriate combinations of

concentrations, but this possibility needs further

filters in the excitation and monitoring systems .

study.

Fluorescence quenching of fluoroquinolone RESULTS AND DISCUSSION

The concentration of KI as quencher was varied from 0 .025 to 0 .2 M, a range in which it has

Influence of pH and polarity of the medium on the absorbance and fluorescence spectra

been shown that iodide concentration induces no changes in lipid bilayer structure [16] . We con-

The methods for the determination of fluoro-

sider that the differences in the intensity of the

quinolone were UV-visible spectrophotometry and spectrofluorimetry . The dissociation of the

emission spectra of fluoroquinolone, either free

carboxyl group in fluoroquinolone produces

ferent environments of the drug, which may af-

changes in the absorption and fluorescence spec-

fect its accessibility to the small molecule of the

tra . The spectra obtained at different pH values

quencher . The Stern-Volmer plots of the iodide

(Fig . 1, top) revealed that the fluoroquinolone

quenching of fluoroquinolone in the absence and

has two absorption bands at 284-272 and 310-340

presence of liposomes deviate from linearity with

nm . At acidic pH the drug shows maximum solu-

upward curvature, indicating the presence of an

bility [2] . In Fig. 1 (bottom), the near-UV spectra

alternative quenching mechanism (static quench-

of fluoroquinolone in different solvents (chloro-

ing) .

or incorporated in liposomes, arise from the dif-

form, ethanol and water) are shown . As can be

Figure 2 presents the data obtained from Eqn .

seen, a decrease in the polarity of the surround-

1 for the free and encapsulated fluoroquinolone

ing medium caused a shift of the absorption

quenched by iodide . The linearity of the KI

bands to longer wavelengths .

quenching allows the calculation of fe values at 25 and 50°C . For free fluoroquinolone, as ex-

In the fluorescence emission spectra (data not shown), as the solvent polarity increased the emission shifted to longer wavelengths . These

pected, almost all the fluorophores are accessible to iodide . Fluorescence quenching studies re-

findings could be indicative of a moderate or low

vealed the existence of two populations of fluo-

affinity of the fluoroquinolones for a hydrophobic

rophores in liposomes, which was in accord with

environment [5] . Red shifts were accompanied by

the partial hydrophobicity of the drugs .

a decrease in the quantum yield of the fluorophore, which also supports this conclusion .

Experiments carried out with fluoroquinolones incorporated in liposomes showed that the fraction of the initial fluorescence that was accessible

Analytical data for fluoroquinolone

to the quencher at 50°C was 76% and that at

Spectral data for fluoroquinolone are given in

25°C was 59% . The higher value obtained above

Table 1 . At the absorption maximum of 278 run,

T. could be interpreted in terms of bilayer fluidity. At 50°C, the bilayer, which is in its liquid-

Beer's law was obeyed in the range 7 X 10-6-



61

MT. Montero et aL /AnaL Chim . Acta 290 (1994) 58-64 .7

.e

.3

.2

.1

0

I

I

I

I

V

I

VAVELENGT OW

\. . WAVELElGTM 4r) Fig . 1 . Top : effect of pH on the fluoroquinolone absorption spectrum : ( ) buffer B ; ( ) buffer C; ( ) buffer A. See text for explanation . Bottom : effect of solvent on fluoroquinolone absorption spectrum : ( ) chloroform ; ( ) ethanol ; ( ) water .



M. T. Montero et aL /Anal . Chim. Acta 290 (1994) 58-64

62 TABLE 1 Analytical characteristics useful for the determination of fluoroquinolone concentration Parameter

UV absorption

Fluorescence

Excitation 278; emission 448 X X 10 -6 1 x 10 -6 -13 x 10 -6 Linear range (M) 7 10 -6-58 Correlation 0 .9983 coefficient 0.9978 7 15 na Wavelength (nm) 278

Number of standard solutions each measured three times .

a

crystal state, permits the accessibility of iodide for the maximum population of fluoroquinolone (both located on the hydrophilic surface and towards the bilayer core) . At 25°C only external fluorophores are exposed to the quencher . It is concluded that the remaining unquenched population (i.e., those fluoroquinolone molecules inac-

cessible to iodide) were either deeper in the liposome bilayers or in the aqueous phase inside the liposome . It is possible also that after removal of the originally soluble fluoroquinolone on the Sephadex column, repartitioning of some quinolone from the hydrophobic bilayer to the external aqueous phase could occur . Such free fluoroquinolone would be accessible to the 1 - . The increased accessibility at 50°C could indicate that the liquid crystalline bilayers more readily allowed partitioning of the fluoroquinolone back into the aqueous phase than did gel state bilayers. Microscope fluorescence showing fluoroquinolone domains in monolayers The visual fields observed under the microscope showed some interesting features . The drug decreased the domain sizes of liquid condensed

9 8 7 6 5 LL

a li

4 3 2 1 0 0

15

30

45

60

75

90

105

1/[KI] (M ) Fig. 2 . Stern-Volmer modified plots of the quenching of fluoroquinolone fluorescence by iodide . Free fluoroquinolone at and (o) 50°C ; fluoroquinolone incorporated in liposomes at (U) 25°C and (A) 50°C.

(0) 25°C

MT Montero et al. /Anal. Chin . Acta 290 (1994) 58-64

63

Fig . 3. Image of the fluoroquinolone DPPC monolayer (0.7 mol DPPC :0.3 mol fluoroquinolone) at a surface pressure of 14 mN M -1 .

lipid in the monolayer . This indicates that quinolone interacted with phospholipids in both the monolayers and bilayers . This is in agreement with the hydrophobic route of permeation of fluoroquinolone into bacteria [9]. From epifluorescence monolayer observations the formation of aggregated drug domains in the lipid environment at higher surface pressures was established (Fig. 3). It still remains unclear if this phenomenon occurs in liposomes, and how it would affect the pharmacological action of the drug. This work was supported by public funds of the University of Barcelona . J. Hernandez-Borrell received grants from the CIRIT (Generalitat

de Catalonia) and by MRC Canada . The authors are indebted to J . Freixas of ITEVE for the fluoroquinolone synthesis .

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