IL NUOVO CIMENTO VOL. 10 D, N. 11 Novembre 1988

Some Structural and Dynamical Properties of Multilamellar Vesicles Incorporated with Propranolol (*)(**).

G. ALBERTINI

Dipartimento di Scienze dei Materiali e della Terra - Ancona, Italia

C. DONATI and F. RUSTICHELLI

Istituto di Fisica Medica - Ancona, Italia

R. S. PHADKE

Chemical Physical, Tara Institute of Fundamental Research - Bombay 400005, India

(ricevuto l'l Agosto 1988)

Summary. - - Spin labelling ESR, calorimetry and ~lp NMR have been used to investigate dipalmitoyl phosphatidylcholine dispersions incorporated with propranolol which is a t~-adrenoceptor blocking drug, widely used in the treatment of arrhythmy. The drug appears to enter the lipid bilayer also at the lowest concentrations and to induce a progressive fluidification of multilamellar vesicles. In fact, the presence of drug reduces the temperature of chain melting transition and the corresponding cooperation number. Furthermore the phase transition L~,- P~, is not observable also at the lowest drug concentrations.

PACS 61.30 - Liquid crystals. PACS 64.70 - Phase equilibria, phase transitions and critical points of specific substances. PACS 87.30 - Biophysics of neurophysiological processes (excluding percep- tion processes and speech).

1. - I n t r o d u c t i o n .

The first step in the absorption of a drug molecule in a living cell involves its interact ion with lipids. The drug initially present in the aqueous solution at the

(*) To speed up publication, the authors of this paper have agreed to not receive the proofs for correction. (**) Work presented at the First USSR-Italy Bilateral Meeting on Liquid Crystals held in Portonovo, Ancona (Italy), September 30-October 2, 1987.

1399

1400 G. ALBERTINI, C. DONATI, F. RUSTICHELLI and R. S. PHADKE

interface with the cell membrane generally dissolves in the lipid matrix, migrates to the other side of the lipid bilayer and enters the aqueous compartment of the cell where it can manifest its action (1). For these reasons, investigations on the lipid-drug interactions are of paramount importance from the point of view of biological as well as liquid-crystal sciences. Moreover, a deep interest has recently arisen for investigations concerning the possibility of vehiculating drugs through carrier liposomes.

Propranolol is a prototype ~-adrenoceptor blocking agent which is widely used in the treatment of arrhythmy, since it exhibits considerable efficacy in the management of cardiac rhythm disorders (~,8). However, its bioavailability is low due to high hepatic extraction ratio or extensive hepatic first-pass metabolism(4). It is, therefore, desirable to encapsulate it in a carrier system which can protect it from immediate biodegradation and deliver it in heart tissue. Lipids are thought to be attractive carrier systems because of their inherent biocompatibility (~). In particular, multilamellar vesicles of dipalmitoyl phosphatidylcholine (DPPC) with or without cholesterol have been considered as carrier systems (6). For these reasons, we have undertaken a detailed study of multilamellar vesicles incorporated with propranolol drug, by using ESR spin labelling, differential scanning calorimetry (DSC) and NMR techniques.

2. - M a t e r i a l s a n d m e t h o d s .

L~ dipalmitoyl phosphatidylcholine (DPPC) was purchased from SIGMA, USA. Spin label 2.2, 6.6-tetramethyl-piperidinyl-N-oxy (TEMPO) was obtained from SYVA. Propranolol (fig. 1) was a gift from CIPLA, INDIA. All other chemicals used are of AnalaR grade.

The vesicles incorporated with the drug were made by using the following procedure. Desired quantities of lipids were dissolved in chloroform. A thin coat of lipid was obtained on the walls of the container by slowly evaporating the solvent under a stream of nitrogen. The residual solvent was evaporated by freeze-drying. Dispersions were made by adding appropriate amounts of buffer

(') Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutic, 2nd edition, edited by G. S. AVERY (Adis Press, Churchill Livingston, 1980). (2) D. T. MASON, A. N. DE MARIA, R. AMSTERDAM, R. ZELIS and R. A. MASSUMI: Drugs, 5, 261, 292 (1973). (8) B. N. SINGH: Drugs, 15, 218 (1978). (4) G. JOHNSON and C. G. REGARDH: Clinical Pharmacokinetics, 1, 233 (1976). (5) A. BIENVENUE and J. PHILIPPOT: Les liposomes: applications th~rapeutiques, edited by F. PUISIEUX and J. DELATBRE (Paris, 1985), p. 149. (~) N. DOUSSET and L. DOUSTE-BLAZY: Les liposomes: applications th~rapeutiques, edited by F. PUISIEUX and J. DELATBRE (Paris, 1985), p. 41.

SOME STRUCTURAL AND DYNAMICAL PROPERTIES ETC. 1401

(10 mM Tris HC1 buffer pH 7.0) or solution of propranolol in buffer, followed by vortexing. The mixture was allowed to equilibrate at 323K for a period of 2 hours before the experimentations. Spin label TEMPO was directly added to the dispersions. The lipid to spin label ratio was maintained to be 100:1 (molar) in all experiments. For NMR experiments 10% 2H~O was used in the buffer and for DSC experiments distilled water, instead of buffer, was used.

+

OCH 2C~CH2NH2CH(CH3) 2

I o. C L -

Fig. 1. - Propranolol.

ESR measurements have been done on a home-made X-band ESR spectrometer with a Varian 12 inch magnet and associated field dial with VFR 2501 magnet power supply. A Varian V-H $601 100 kHz field modulation and detection unit was used. Temperatures to an accuracy of + 1K were measured by using copper-constantan thermocouple placed in close proximity of the sample.

sip NMR experiments were carried out on Bruker AM-500 FT-NMR spectrometer interfaced with Aspect 3000 computer. A broad-band proton decoupling (3 dB) has been employed in all the experiments. A relaxation delay of 1.5s has been used.

Calorimetric curves were obtained using Perkin Elmer calorimeter model DSC-2C with associated data processor. The scan rate was 2.5~ -1. Alumin- ium containers of 20 ~1 capacity were used as sample holders.

3. - R e s u l t s a n d d i s c u s s i o n s .

ESR spin labelling. Spin labels are molecules possessing an unpaired electron. A typical spectrum of freely tumbling spin label in magnetic field consists of there sharp lines (7) (fig. 2a)). It is customary to record the first derivative of absorption lines. Double integration can yield the spin concentration in a particular state. The parameters which can be studied are the resonance field, the hyperfine splitting and the spin concentration. If the line widths do not change over the range of temperatures of experimentation, one

(7) E . J . SHIMSHICK and H. M. MC CONNEL: Biochemistry, 12, 2351 (1973).

1402 G. ALBERTINI, C. DONATI, F. RUSTICHELL! and R. S. PHADKE

can use the line heights as a parameter for estimating the spin concentrations. The spin label TEMPO possesses the special property of partitioning into the

water as well as lipid phase (in our case, the relative partitioning at room temperatures for 100 mM lipid is 50:1 molar ratio). The extent of partitioning depends upon the fluidity of the lipid and hence this spin label is popularly employed to study the phase transition characteristics(S). At 9.5GHz the spectrum of TEMPO in multilamellar vesicles exhibits two-component character in the high-field resonance line (fig. 2b)). Figure 2c) reports the spectrum obtained after incorporation of propranolol at a molar ratio of drug to DPPC = 1/5.

The two components originate from i) TEMPO dissolved in the lipid phase (H) and ii) that dissolved in the aqueous phase (P). The relative partitioning in the

N

i i Fig. 2. - X-band ESR spectrum of spin label TEMPO: a) acqueus solution, b) DPPC liposomes, c) DPPC liposomes incorporated with propranolol. (Propranolol/DPPC molar ratio r = 0.2.)

(8) R. S. PHADKE, N. V. KUMAR, R. V. MOSUR, A. SARAM and G. GOVIL: Int. J. Quantum Chem., 20, 85 (1981).

SOME STRUCTURAL AND DYNAMICAL PROPERTIES ETC. 1403

two phases varies with temperature. A dimensionless parameter considering the line heights f = H / ( H + P ) can be used to monitor the phase transition characteristics of the system; fig. 3 depicts a plot o f f vs. temperature. The curve for pure DPPC vesicles exhibits a sigmoidal character with abrupt changes in f values corresponding to pretransition (~ 307K) and main transition (= 314K). The presence of propranolol has altered the curve considerably. Firstly the pretransition is no more distinguishable. Only one sudden jump in f value is evident which takes place at much lower temperature as compared to DPPC vesicles. The retaining of sigmoidal nature of the phase transition curve indicates that the cooperative nature of the phase transition persists even after incorporation of large amounts of propranolol (drug: DPPC = 1 : 5 molar).

0.6

0,2

I I

300 3tO Y;empere~ture ( K )

b)/a) I I I I

320 330

Fig. 3. - Variation of ((f)~ with temperature at pH = 7.0. a) �9 DPPC (100mM), b) • DPPC (100 raM) propranolol (20 mM).

Differential scanning calorimetry (DSC). Calorimetry offers as a sensitive tool to study phase transition behaviour (9-11). A careful investigation of the behaviour of DPPC multilamellar vesicles incorporated with propranolol has been done using calorimetric methods. Figure 4 shows calorimetric scans for vesicles obtained by using DPPC and water in weight ratio 1/7 and containing propranolol in different drug to lipid molar ratios (r). For pure DPPC vesicles (r= 0), the calorimetric scans exhibit two peaks corresponding to the first endothermic transition (from L~, to P~, phase) and to the second endothermic

(9) D. CHAPMAN, P. M. WILLIAMS and B. D. LADBROOKE: Chem. Phys. Lipids, 1, 445 (1967). (Io) R. N. MC ELHANEY: Chem. Phys. Lipids, 30, 229 (1982). (11) S. MABREY and S. M. STURTEVANT: Proc. Nat. Acad. Sci USA, 73, 3867 (1976).

1 4 0 4 G. ALBERTINI, C. DONATI, F. RUSTICHELLI and R. S. PHADKE

i I I I ^ I 'l I

L,, P~' / \ '~

o~) r = O

b) r = 0.05

n :O .20

I I I I i 300 305 310 315 320

tempepabtur'e (K)

Fig. 4. - Calorimetric scans for DPPC vesicles containing different concentrations of proprano]o] ( r=propranolo l /DPPC molar ratio): a) r = 0, b) r = 0.05, c) r = 0.2. The healing rate was 2.5K per min.; DPPC/water ratio was 1/7 (w.w.).

transition (from P~, to L~ phase) which are characteristics of the lipid. On incorporation of propanolol the character of the curve alters considerably. In fact, the first calorimetric peak is not detectable even for the smallest propranolol concentration (r = 103). The ESR data (fig. 3) did suggest such an effect but did not give the definite evidence. Moreover, higher propranolol concentration imparts a broad feature to the main transition peak, in agreement with the increasing of the temperature interval of transition observed by ESR. On further increasing the propranolol content (r = 0.2) the phase transition not only broadens but shifts considerably towards lower temperatures. A detailed study of the effect of the presence of increasing amounts of propranolol on the chain melting temperature of the lipid vesicles is shown in fig. 5. For small amounts of th~ drug r < 5.10 .2 there is no significant change in the transition

SOME STRUCTURAL AND DYNAMICAL PROPERTIES ETC. 1405

315.0

312.5

~ 310.0

E ~ 307.5

305.0 t i / / / / i L i i i i i i i I I i I i i i i i i i i i l J l l l I - -

0 10 -3 10 -2 10 -1 moLo~r ro~t/o r

Fig. 5. - Temperature of the intense calorimetric peak v s . the propranolol / DPPC molar ratio r. DPPC/water ratio = 1/7 (w.c.).

temperature, but further increase in r causes lowering of transition temperature.

A greater insight can be obtained by plotting the width of the transition peak against temperature (fig. 6). By increasing incorporation of propranolol, the broadening of calorimetric peak increases too; this indicates that the presence of propranolol affects cooperation. The cooperation number (CN) can be calculated (10) from the expression

4RT 2 C N = - -

A T , A H '

where R is the gas constant, T is the transition peak temperature, AT is the full width at half-maximum and 5/-/is the transition enthalpy. CN decreases with

~3

~2

Z,'

L I # l I I I I J l l l

0 10 -3

1,t [ i i I i 1 1 I i i i i i i i I

10 -2 10 -1

rno~obr ro~t io r

Fig. 6. - Full width and half-maximum of the intense calorimetric peak v s . the propranolol/DPPC molar ratio r.

1406 G. ALBERTINI, C. DONATI, F. RUSTICHELLI and R. S. PHADKE

increasing incorporation of propranolol drug and attains a constant level after propranolol/DPPC molar ratio of ~ 4 .10 -2 (fig. 7). This can be attributed to the increase in the freedom of the lateral motion of lipid molecules consequent to the fluidifying effect of propranolol.

75

c..

so

o

~2s

10 -3 J , i l t L l l L I J L l l l l l l

10-2 10-I mo~o~r" re, r i o r"

Fig. 7. - Cooperation number CN vs . the propranolol/DPPC molar ratio r.

31pNMR. Nuclear magnetic resonance is unique as regards its non- destructive, noninvasive character (12). For phospholipid dispersions 81p offers as a natural, nonperturbing probe on account of its 100% natural abundance. A typical ~lp NMR spectrum depends upon the electronic-charge distribution around the phosphorus nucleus and its orientation with respect to the applied magnetic field. In the case of the multilamellar vesicles, all orientations are equally probable. This attributes a powder pattern character to the NMR spectrum (fig. 8). The spectrum consists of an intense peak separated from a broad shoulder(l~,"). The separation, i . e . chemical shift anisotropy (CSA), is of the order of 48p.p.m. at 306K for DPPC 100raM (fig. 8a).

Additional contributing factors originate from the motions: i) Brownian tumbling of vesicles, ii) rotation of the lipid around its long axis, iii) segmental motion about chemical bonds. The motions average the chemical shift anisotropy resulting in bringing peaks closer as the temperature is increased (fig. 8b)).

The presence of propranolol is seen to bring a further narrowing of the signal (fig. 8c)). The signal consists of a single sharp peak of width of the order of lp.p.m. This indicates that the phosphorus is now experiencing structurally

(12) j . SEELIG: Biochim. Biophys. Acta, 515, 105 (1978). (13) R. BRASSEUR, V. CABIAUX, J. A. KILLIAN, B. DE KRUIJFF and J. M. RUYSSCHAERT: Biochim. Biophys. Acta, 855, 317 (1986). (14) S. SRIVASTAVA, R. S. PHADKE and G. GOVIL: submitted.

SOME STRUCTURAL AND DYNAMICAL PROPERTIES ETC. 1407

D PPC DPPC +pr 'o lapcbnol , oi.,

T= 323 K / A~X b)

H PPM I I I I L I I I I I I

- z~o - 2 0 0 20 4.0 H PPM

Fig. 8. - 31p NMR chemical shift anisotropy (CSA) for DPPC multilamellar vesicles; a) DPPC (100 mM) at 306 K, line broadening of 20 Hz; b) DPPC (100 mM) at 323 K, line broadening of 20 Hz; c) DPPC (100 mM) and propranolol (20 mM) at 323 K, line broadening of 10 Hz.

symmetric and/or motionally averaged isotropic situation. This can be attributed to the fluidizing effect of the drug which is in accordance with spin labelling ESR and DSC observations.

4. - Conclusion.

ESR spin labelling, NMR spectroscopy and calorimetry indicate that the incorporation of propranolol drug induces fluidization in the multilamellar vesicle of DPPC and melting of lipid hydrocarbon chains at lower temperatures as compared to pure lipid dispersions. Moreover, the phase transition L~,-o P~, is not observable even at the lowest drug concentration suggesting a penetration of propranolol molecules inside the lipid bilayers also for the lowest concentrations. Finally the cooperation number is progressively reduced as far as the drug concentration increases.

It is a pleasure to thank Prof. M. Signorino for useful discussions. One of the authors (R. S. Phadke) has carried out part of this work with the support of the (,ICTP Programme for Training and Research in Italian Laboratories, Trieste, Italy)). The facilities provided by 500 MHz FT NMR National Facility, supported by the Department of Science and Technology and located at T.I.F.R., Bombay, India, are gratefully acknowledged. Prof. B. Venkatraman and Mr. V. R. Bhagat are gratefully acknowledged for ESR facilities.

This work was supported by Regione Marche. Part of the equipment was obtained on the basis of financial help from CNR and MPI.

1408 G. ALBERTINI, C. DONATI, F. RUSTICHELLI and R. S. PHADKE

�9 R I A S S U N T 0

Liposomi di dipalmitoillecitina contenenti propranololo, un beta-bloccante ampiamente utilizzato contro le ari tmie cardiache, sono stat i studiati utilizzando tecniche di risonanza magnetica elettronica con marcatori di spin, tecniche di risonanza magnetica nucleare sul fosforo 31 e tecniche di calorimetria. I1 farmaco anche alle pifi basse concentrazioni pene t ra nel doppio s trato lipidico e a concentrazioni maggiori eserci ta un sensibile effetto fluidificante. Infat t i la presenza del propranololo induce un abbassamento nella t empera tu ra di fusione delle catene alifatiche e fa diminuire la cooperativit~ della corr ispondente transizione. Inoltre la transizione di fase L z , - Pz, non ~ piti rivelabile anche alle piti basse concentrazioni di farmaco.

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