Luminescence Properties of Liposomes Incorporating Two Kinds of

Nov 1, 1993 - Kinds of Cyanine Dyes: Excitation Energy Transfer ... Liposomes incorporating two kinds of J-aggregates at the same surface were prepare...
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Langmuir 1993,9, 3395-3401

3395

Luminescence Properties of Liposomes Incorporating Two Kinds of Cyanine Dyes: Excitation Energy Transfer between J-Aggregates Tomoo Sato,* Mitsunori Kurahashi, and Yoshiro Yonezawa Division of Molecular Engineering, Graduate School of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-01, Japan Received May 4,1993. In Final Form: August 23,199P Liposomes incorporatingtwo kinds of J-aggregates at the same surface were prepared by the adsorption method. Excitation energy transfer between the J-aggregatescaused quenching of resonance fluorescence of donor aggregate and sensitization of resonance fluorescence of acceptor aggregate. It has been shown that the J-aggregate of l,lf-diethyl-2,2’-cyaninechloride and 5,6:5’,6’-dibenzo-l,l’-diethyl-2,2’-cyanine chloride behave like the energy donor and the J-aggregate of 5,5‘-dichloro-3,3‘-diethyl-9-phenylthiacarbocyanine chloride and 3,3’-dimethyl-9-pheny1-4,5:4‘,5’-naphthothiacarbocyanine chloride behave like the energy acceptor. The dependence of quenching and sensitization efficiencies on the composition of two cyanine dyes obeyed an empirical Perrin relation. It is proposed that each kind of dye independently forms the J-aggregate which consists of about 20 monomer units and that the J-aggregates are distributed at random at the liposomal surface as if each of them was a large supermolecule. 1. Introduction

Excitation energy transfer and energy trapping in molecular assemblies, such as micelles, liposomes, and monolayers, have received much attention1 in connection with photobiologicalproce~ses.~*~ Energy transfer between donor and acceptor molecules in the molecular assemblies has been analyzed in terms of Forster’s dipole-dipole t h e ~ r y . ~Most J j works in this field have been done by using donor and acceptor molecules in the monomeric form. However, energy transfer between molecular aggregates sometimes plays an important role in light-harvesting systems of photosynthesis.M Some cyanine dyes can form the J-aggregate, characterized by a sharp J-band and resonance fluorescence, in aqueous solutions in the presence of certain electrolytes?JO The J-aggregate is the molecular cluster characterized by the small angle between the transition moments and these centersl1-l3 and has been used for spectral sensitizer in silver halide photography.14JS We have recently proposed @Abstractpublished in Advance ACS Abstracts, November 1, 1993. (1)Yamazaki, I.; Tamai, N.; Yamazaki, T. J. Phys. Chem. 1990,94, 516. (2)Amesz, J. Photosynthesis: New Comprehensive Biochemistry; Elsevier: Amsterdam, 1987;Vol. 15. (3)Scheer, H.,Schneider,S., E&. Photosynthetic Light Harvesting Systems. Organization and Function; de Gruyter: Berlin, 1988. (4) Fijrster, Th. Dhcws. Faraday SOC.1959,27,7. (5)Agranovich, V. M.; Galanin, M. D. Electronic Excitation Energy Transfer in Condensed Matter; North-Holland New York, 1982. (6)Cawgrove,T. P.;Yang, S.; Struve, W. S. J.Phys. Chem. 1988,92, 6121;J. Phys. Chem. 1988,92,6790. (7)Beck, W. F.;Sauer, K.J. Phys. Chem. 1992,96,4658. (8)Mimuro, M.; Nozawa, T.; Tamai, N.; Shimada, K.;Yamazaki, I.; Lin,S.; Knox, R. S.; Wittmershaw, B. P.; Brune, D. C.; Blankenship, R. E.J. Phys. Chem. 1989,93,7503. (9)Jelley, E.E. Nature (London) 1936,138,1009. (10)Scheibe, G. Angew. Chem. 1936,49,563. (11)Emerson, E. S.;Conlin, M. A.; Rosenoff, A. E.; Norland, K. S.; Rodriguez, H.; Chin, D.; Bird, G. R. J. Phys. Chem. 1967,71,2396. (12)(a) Czikkely, V.;Fijrsterling, H. D.; Kuhn,H. Chem. Phys. Lett. 1970,6,11.(b)Biicher, H.; Kuhn,H. Chem. Phys. Lett. 1970,6,183.(c) Czikkely, V.; Fijrsterling, H. D.; Kuhn,H. Chem. Phys. Lett. 1970,6,207. (13)Sturmer,D.M.; Heseltine,D. W. The Theory ofthe Photographic Processes, 4th ed.; James, T. H., Ed.; MacMillan: New York, 1977;p 194. (14) West, W.; Gilman, P. B., Jr. The Theory of the Photographic Processes, 4thed.; James, T. H., Ed.; MacMillan: New York, 1977;p 251. (15)Steiger, R.; Zbiinden, F. J. Imaging Sci. 1988,32,64.

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Figure 1. Structural formulas of cyanine dyes. a tractable method to prepare the J-aggregate at the surface of liposomes containing acidic phospholipids.16 In the previous reports, we managed to prepare the liposomes having two kinds of J-aggregates at the surface.16J7 Resonance fluorescence of one aggregate (donor) was quenched and resonance fluorescence of another aggregate (acceptor) was sensitized. The advantages of the liposomal solutions composed of small unilamellar vesicles (SUV)are that the dye aggregatesare easily formed even in a dilute solution and that direct spectroscopic measurements are possible because of relatively low light scattering. In the present study, we have examined excitation energy transfer between the donor and the acceptor aggregates and discussed the structure and distribution of these aggregates at the liposomal surface. 2. Experimental methods Structuralformulas of four cyanine dyes used in this work are shown in Figure 1. The cyanine dyes l,lf-diethyl-2,2’-cyanine chloride (dye-I),5,65’,6’-dibenzo-1,1f-diethyl-2,2’-cyanine chloride (dye-11),5,5’-dichloro-3,3’-diethyl-9-phenylthiacar~anine chloride (dye-111), and 3,3’-dimethy1-9-phenyl-4,54’,5’-naphthothiacarbocyanine chloride (dye-IV) were purchased from Nippon Kanko Shikiso Co. L-a-Dimyristoylphosphatidylcholine (DMPC) and dicetyl phosphate (DCP) were purchased from (16)Sato, T.; Yonezawa, Y.; Hada, H. J. Phys. Chem. 1989,93,14. (17)Sato, T.;Yonezawa, Y.; Kurokawa, H.; Kurahashi, M.; Wada, Y.; Tanaka, T. Thin Solid Films 1992,210l211,172.

0743-7463/9312409-3395$04.0010 0 1993 American Chemical Society

3396 Langmuir, Vol. 9, No. 12, 1993 Sigma and Nacalai Tesque Co., respectively. Singly distilled water was used throughout the work. Liposomeswere prepared from the suspensionof phospholipids (DMPC and DCP, molar ratio 3:l-1:l) by use of a probe type sonicator as described before.16 The chloroform solution of DMPC and DCP in a round-bottomflask was slowly evaporated with the aid of a rotary evaporator on a water bath at 40-70 "C to remove the solvent. Thin films of phospholipids at the flask surface were hydrated with the singly distilled water at 40-70 "C and the mixture was shaken vigorously. The dispersion were sonicated at 40-70 O C under a stream of nitrogen by use of a Nissei US-300 sonifier equippedwith a 264 probe (Nippon Seiki Seisakusho Co.) at 300 W output for 30-60 min. The concentration of phospholipids, [DMPC + DCP] was 1.6 X 1V M (1 M = 1 mol dm-9). The pH of the solution was adjusted to 6.0 with 0.1 M NaOH. The liposomes, prepared in this manner, were SUV, whose diameter was estimated to be 30-40 nm.18JD The liposomal solution was added to the 1odM dye-I1solution in HZO/CHsOH (99.7:0.3 (v/v))until [DMPC + DCP] = 9.1 X 1od M. After completion of the dye-I1 aggregation, a proper amount of aqueous solution of 1o-L M dye-I11was added. In this manner, we could prepare the dye-IIdye-I11liposome, which adsorbed J-aggregatesof dye-I1and those of dye-I11at the same surface.16 The molar ratio of dye-I11to dye-I1was controlled by changing the amount of dye-I11 solution. Absorption spectra and fluorescence spectra of the liposomal solutions in the quartz cell (10 x 10 X 45 "3) were recorded on a Hitachi 200'-20 spectrophotometer and a Hitachi MPF-4 spectrofluorometer,respectively. The excitation wavelength (Ll for the fluorescence measurements was 450 nm unless otherwise stated. The spectraldistributionof the sensitivity of the detection unit of MPF-4 was corrected. All measurements were carried out in air at room temperature. 3. Results 3.1. Absorption and Luminescence Properties of Liposomes Incorporating One Kind of J-Aggregate. We at first prepared the liposomes incorporating one kind of cyanine dye. The absorption and fluorescence spectra of the 106 M dye-I1 solution in the presence or absence of the DMPC:DCP = 1:l liposomes are shown in Figure 2A. In the absence of liposomes, a monomer band (Mband) at X = 553 nm and a dimer band (D-band) a t X = 508 nm were seen in the absorption spectrum but monomer fluorescence was not observed in the fluorescence spectrum. Addition of liposomesto the solution brought about an appearance of a strong J-band at X = 598-600 nm, together with resonance fluorescence at X = 606-608 nm, indicating the formation of dye-I1 aggregate (dye-I1 liposome). Meanwhile, the M-band and the D-band were decreased. Adsorption of dye-I1 at the liposomal surface would be caused by electrostatic interaction between the positive charge of dye-I1 and the negative charge of DCP, resulting in the formation of J-aggregate. The molar absorptivity, e, of the dye-I1 aggregate per dye monomer was 1.74 X lo5 M-l cm-l at X = 598-600 nm. Figure 2B shows the absorption and fluorescencespectra of the 10-6 M solution of dye-I11in the presence or absence of the DMPC:DCP = 1:l liposomes. The absorption spectrum of the dye-I11solution had a M-band a t X = 562 nmandaD-bandat X = 518nmin theabsenceofliposomes. Monomer fluorescence was observed a t X = 588 nm, but the spectrum is not given in Figure 2B for simplicity. Immediately after adding liposomesto the dye-I11solution, the M-band and the D-band decreased and a J-band at X = 659-665 nm (t = 1.54 X 106 M-1 cm-1) developed (dye(18) (a) Huang, C. Biochemistry 1969,8,344. (b) Hauser, H.; Phillips, M. C.; Stubbs, M. Nature 1972,239, 342. (19) Sato, T.; Kijima, M.; Shiga, Y.; Yonezawa, Y. Langmuir 1991, 7 , 2330.

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Wavelength (nm) Figure2. Absorption (solidlines) and fluorescence (brokenlines) spectra of dye-I1 solution (A) and dye-I11 solution (B)in the presence or absence of the DMPC:DCP = 1:l liposomes: (A) a, 0.33 vol % CHaOH/HzO solution of dye-I1 (1.0 X lod M); b, c, 0.31 vol % CHsOH/HzO solution of dye-I1 (9.4 X 10-8 M) and liposome ([DMPC+ DCP] = 9.1 X 1odM);(B)a, aqueoussolution of dye-I11 (1.0 X 1od M); b, c, aqueous solution of dye-I11 (9.4 X 10-8 M) and liposome ([DMPC + DCP] = 9.1 X 1od M). Excitation wavelength )b. = 450 nm.

I11 liposome). Resonance fluorescence was observed at X = 670-675 nm. 3.2. Absorption and Luminescence Properties of Liposomes IncorporatingTwo Kinds of J-Aggregates. We prepared the dye-IIdye-I11 liposomes incorporating dye-I1 and dye-I11 at the surface. The 10-4 M dye-I11 solution was added to the liposomal solution incorporating the dye-I1 aggregates, until [dye-1111 = 8.6 X 1o-S M. The absorption and fluorescence spectra of the liposomal solution are shown in Figure 3A. The absorption spectrum of the dye-Ikdye-I11 liposome was nearly equal to the superposition of the absorption spectra of the dye-I1 liposome and the dye-I11 liposome, apart from a small change in the ratio of the absorbance at the J-bands. The absorption peaks at X = 598-600 nm and X = 652-658 nm correspond to the J-bands of the dye-I1 and the dye-I11 aggregates, respectively. This observation indicates that dye-I1 and dye-I11 form the J-aggregates independently at the liposomal surface. If dye-I1 and dye-I11 formed a single J-aggregate like amalgamation type mixed c r y ~ t a l s , ~ a new J-band should appear. In the fluorescence spectrum, resonance fluorescence a t X = 608 nm and 665-670 nm arose from the dye-I1 and the dye-I11aggregates, respectively. However, compared with the fluorescence spectra in Figure 2, the fluorescence intensity of the dye-I1 aggregate decreased by a factor of 10 and that of the dye-I11 aggregate increased about 3 (20) Onodera, Y.; Toyozawa, Y. J. Phys. SOC.Jpn. 1968,24,341.

Luminescence Properties of Liposomes

Wavelength ( n m )

1-5.1

kfI

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3.3. Dependence of Energy Transfer Efficiency on Surface Density of Acceptor. The absorbance at the J-band and the apparent intensity of resonance fluorescence of the donor (dye-11)aggregate, Ad and Id, and those of the acceptor (dye-111)aggregate,A, and ia,were obtained from the absorption and fluorescence spectra of the dyeIIdye-I11 liposomes. Ado and Ido denote the absorbance and fluorescence intensity of the donor aggregate in the absence of acceptor aggregate (dye-I1liposome), and Aao and iao those of the acceptor aggregate without donor aggregate (dye-I11liposome). A correction of the reabsorption of donor fluorescencewas not taken into account, because the concentration of the donor aggregate was almost constant in the dye-1I:dye-I11 liposomes. The reabsorption of acceptor fluorescence was corrected by using the followingrelation between iao andAao,as observed in dye-I11 liposomes.

i," = B(1- exp(-CA,")j (1) Here, the fitting parameters B and C were 4.37 and 1.60, respectively. The corrected fluorescence intensity of the acceptor aggregate in the absence and the presence of the donor aggregate, Iao and I,, were given by

:I = I, =

Wavelength ( n m )

Figure3. Absorption (A,a) and fluorescence (A, b; B, a-f) spectra of dye-IIdye-I11liposomes. Excitation wavelength Lx= 450 nm. The scale of fluorescence intensity is the same as that in Figure 2. (A) 0.29 vol % CHsOH/H20 solution of dye-II(8.6 X 106 M), dye-III(8.6 X 106 M), and DMPC:DCP = 1:l liposome ([DMPC + DCP] = 8.3 X 1od M). (B)Variations of the fluorescence spectra by changing the composition of dye-IIdye-I11 (a) [dyeI11 = 9.4 X 1o-S M, [dye-1111 = 0.0 X 10-8 M, [DMPC + DCPI = 9.1 X 1od M; (b) [dye-111 = 9.3 X 1o-S M, [dye-1111 = 1.0 x 1o-S M, [DMPC + DCPI = 9.0 X 1 P M, (c) [dye-I11 = 9.2 X 1o-S M, [dye-1111 = 1.9 X 1o-S M, [DMPC + DCPI = 8.9 x 106 M; (d) [dye-I11 = 9.1 X 1o-S M, [dye-1111 = 2.8 X 1o-B M, [DMPC + DCPI = 8.8 X 1od M (e) [dye-I11 = 8.9 X 1o-S M, [dye-1111 = 4.5 X 1o-S M, [DMPC + DCPI = 8.6 X 106 M, (f) [dye-I11 = 8.6 X 1o-S M, [dye-1111 = 8.6 X 1o-S M, [DMPC + DCPI = 8.3 X 1od M.

times. Quenching of resonance fluorescenceof the dye-I1 aggregateand sensitization of that of the dye411aggregate would be caused by excitation energy transfer from the dye-I1aggregate to the dye-111aggregate. In this case, the dye-I1aggregate and the dye-I11aggregate behave like the energy donor and the energy acceptor, respectively. It is likelythat efficiency of energy transfer would change with the composition of donor and acceptor a t the liposomal surface. We prepared the dye-1I:dye-I11liposomes with various molar ratios and measured the absorption and fluorescence spectra. The variations of the fluorescence spectra with addition of the 10-4M dye-I11 solution to the dye-I1 liposomes are shown in Figure 3B. The addition of the dye-I11 solution caused the decrease of resonance fluorescence of the dye-I1 aggregate and the increaseof resonance fluorescence of the dye-I11aggregate. Although the amount of dye411 for line f was twice as large as that for line e in Figure 3B,resonance fluorescence of the dye-I11aggregate did not increase much. Therefore, reabsorption of resonance fluorescence by the dye-I11 aggregate had a seriouseffect on the fluorescence intensity.

1.60A: 1- exp(-l.GOA:)

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The intensity of sensitized fluorescence itself, AI,, of the acceptor aggregate was defined by

(4) In a conventional Perrin kinetics of energy transfer, the corrected ratio is expressed by2lPz2

where r ( A ) denotes the surface density of the acceptor and Sthe "Perrin volume". We could expect the following relation between I d and AI,. = I d + (l/S) Ma

I:(Ad/A:)

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(7) Here, 9 denotes a ratio of the apparent fluorescence quantum yields of the acceptor without donor, 4a, to that of the donor without acceptor, 1 = d,/&d

(8)

4 was estimated from the fluorescence intensity per absorbanceat the excitation wavelength. The fluorescence intensity of acceptor was corrected with eq 2. In the dyeIIdye-I11 liposomes, 17 was 3.8. We have introduced two quantities, HIand Hz. HI = Id ~

~~

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(21)Perrin, F. C.R.Hebd. Seances Acad. Sci. 1924,178,1978. (22)Inokuti, M.;Hirayama, F. J. Chem. Phys. 1966,43, 1978.

3398 Langmuir, Vol. 9,No. 12,1993

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From eqs 5, 7,9, and 10

3

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ln(H1) and ln(1 + Hz) are termed quenching coefficient and sensitization coefficient, respectively. ln(H1) and ln(1 H2) versus r(A) plots should fit to a single line passing the original point with a slope S. The total number of dye-I11 molecules, which form J-aggregate at the liposomal surface, N (dm-3)is given by (Ad4 NA. Here, NA is Avogadro's constant. The area of the outer surface of the liposome, A(nm2dm-3),is equal to (2/3)(0.5[DMPC DCPINA) under the assumptions that the outer surface of the liposome is twice that of the inner surface and that the surface area of the phospholipid molecule is 0.5 nm2. The surface density of the acceptor at the liposomal surface, r(A)(nm-2) is then given by

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r ( A ) (nm-2) = 3(Aa/e)/[DMPC + DCPI (12) ln(H1) versus Udye-111) plot (the quenching plot) and ln(1 + H z ) versus r(dye-111) plot (the sensitization plot) are given in Figure 4A. Both plots could not fit a single straight line passing the original point. Each curve seems to consist of two straight lines. One is a steep line in the smaller surface density region and another is a gentle line not passing the original point, which dominates in the large surface density region. Furthermore, the slopes of two straight lines for the quenching and sensitization plots are not equal. To express our results in the form of eq 11, we must introduce the trap state as described in the discussion section. 4. Discussion 4.1. Structures of Two Kinds of J-Aggregates Adsorbed at the Liposomal Surface. In excitation energy transfer, quenching of donor fluorescence is usually accompanied with sensitization of acceptor fluorescence. An inspection of Figure 3B reveals that sensitization of acceptor fluorescence was not so evident in the presence of a small amount of the acceptor molecules in spite of much decrease of donor fluorescence. Such a result is evident in the two curves in Figure 4A. Although the sensitization coefficient is almost zero for r (dye-111) less than 0.05 nm-2, the quenching coefficient is more than 1. We now plot the sensitization coefficient,ln(1+ Hz) versus the quenching coefficient, ln(H1) (Figure 4B). This plot yields a straight line, whose intercept at the ln(H1) axis, Xint,is 1.6. It means that a part of fluorescence quenching of the donor aggregate proceeds without having any relation to energy transfer to the acceptor aggregate. To account for the results, we have introduced a certain defect state, 2, formed as a result of addition of the dyeI11solution to the dye-I1liposomes. 2 is either the dye-I11 monomer or the imperfection in dye-I1 aggregate caused by dye-111. However, the amount of 2 seems to be too low to be detected by conventional methods. The properties of 2 are assumed as follows. (1)2 is formed when the concentration of dye-I11is too low to form the J-aggregate. (2) After J-aggregation of dye-I11 starts, the amount of 2 remains constant irrespective of the concentration of dye111. (3) The excited state of 2 is easily deactivated by nonradiative processes. When the concentration of dyeI11 is not so high, a part of the resonance fluorescence of the donor aggregate is quenched by 2, while sensitization of resonance fluorescence of the acceptor aggregate does

In(H1)

Figure 4. Perrin type plots for quenching and sensitizationof resonance fluorescence of dye-1I:dye-I11 liposomes (A) and dependence of the sensitization coefficient on the quenching coefficient (B). Reabsorption is corrected. (A) Dependence of ln(H1) (a, X) and ln(1 + Hz)(b, 0)on the surface density of dye-111,IYdye-111). (B)Before (a, 0 )and after (b, 0)correction of Ido and 4d, according to eqs 13 and 14 in the text.

not occur. Quenching of donor fluorescence by 2 causes a nonzero Xint in Figure 4B. To analyze energy transfer between the donor and the acceptor aggregates, new I d o and f$d, which .take the presence of 2 into account, should be introduced. We define the corrected Ido, Ido (new), and quantum yield, f$d(new),by the following equations. I:(new)

= 12/exp(Xh,)

(13)

(14) f$d(new)= f$,/exp(Xht) Then, the ratio of quantum yield, q(new), is given by dnew) = rl exp(Xht) (15) ln(H1) and ln(1 + H2) are calculated again by using Ido(new) and ?(new). We plot ln(1 + Hz) versus ln(H1) in Figure 4B. A new plot yields a straight line passing through the original point with the slope of 1. Corrected quenching and sensitization plots are given in Figure 5A. Both plots give the common straight line passing through the original point with the slope of 2.8 nm2. In the Perrin type k i n e t i ~ s , ~energy l - ~ ~ transfer takes place only when the acceptor molecule lays within the circle of the radius Ro, the center of which is occupied with the donor molecule. The area of the circle, S ("Perrin volume"; &02) appears in eq 11. If one acceptor aggregate is composed of n monomer units on an average, the surface density of the acceptor aggregate, I?(&), is equal to r(A) divided by n.25 Therefore, the slope of the quenching and sensitization plots for the acceptor aggregate is n times larger than that in Figure 5A. In addition, we should note that the donor aggregate and the acceptor aggregate themselves occupy certain domains, i.e., a sort of "excluded volume" in the surface area.25 If each donor and acceptor aggregate is a disk (23) Chandrasekhar, S. Reu. Mod. Phys. 1943,15,1. (24) Boulu, L. G.; Kozak, J. J. Mol. Phys. 1988,65, 193. (25) Yonezawa, Y.; Kurokawa, H.; Sato, T. J. Lumin. 1993,54, 285.

Luminescence Properties of Liposomes

Langmuir, Vol. 9,No. 12,1993 3399

f

0 - 0 0

0.2

0.4

0

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r

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Figure 5. Perrin type plots for dye-IIdye-I11liposomes (A) and dye-1I:dye-IV liposomes (B)after the correction of Ido and t$d. Reabsorption is corrected. (A) Dependence of ln(H1) (x) and on the surface density of dye-111, r'(dye-111).(B) ln(1 + Hz)(0) Dependence of ln(Hd (X) and ln(1 + Hz)(0)on the surface density of dye-IV, I'(dye-IV).

composed of n monomer units whose area is 0.6 nm2 per one molecule, the occupied area of one aggregate with the radius r, is m-2 = 0.6n. Because of the excluded volume, each aggregate cannot approach each other less than 2r. A "Perrin volume" is then approximated by r(Ro2+ 4rRo), which is the area of a ring region, whose radius is larger than 2r and smaller than 2r + Ro. Then eq 11for energy transfer between J-aggregates is reduced to ln(H,) = ln(1 + H2) = T(R:

+ 4rR0)I'(A)/n

(16)

From the slopes of the quenching and sensitization plots for dye-1I:dye-I11 liposomes and Ro of about 2 nm,26 the number of monomer units forming J-aggregate turns out to be 20. This value is not so far from the value estimated from the spectral shift and band width of the J-band.27-29 A structural model of the J-aggregates of two kinds of cyanine dyes adsorbed at the liposomal surface is shown in Figure 6 . The observation of the two J-bands in the absorption spectra suggests that each kind of dye molecule independently forms the J-aggregates. According to Kuhn et al.,12 we adopted the brick-stone arrangement as the structure of a J-aggregate. The number of monomer units consisting of one J-aggregate is about 20. Each J-aggregate distributes randomly at the liposomal surface with the distance between them being close enough to allow energy transfer between the J-aggregates. The diameter of the aggregates, r, of ca. 3.9 nm is larger than the critical radius, Ro. As the size of the donor and acceptor aggregates is rather larger than the average distance between them, Fbrster-type analysis of the energy transfer between the J-aggregates would be not so straightforward. Therefore, we have tentatively interpreted our results on the basis of the empirical Perrin kinetics as a crude first approximation. 4.2. Excitation Energy Transfer between Other Combinations of J-Aggregates. We have examined (26) Yonezawa, Y.; Hayashi, T. J. Lumin. 1990,47, 49. (27) Zuckerman, B.; Mingace, H. J. Chem. Phys. 1969,50, 3432. (28) Knapp, E. W . Chem. Phys. 1984,85, 73. (29) (a) Kemnitz, K.; Yoshihara, K.; Tani, T. J. Chem. Phys. 1990,94, 3099. (b) Tani, T.; Suzumoto T.; Kemnitz, K.; Yoshihara, K. J.Phys. Chem. 1992,96,2178.

Figure 6. Schematic representationof J-aggregates of two kinds of cyanine dyes adsorbed at the liposomal surface. Each kind of dye molecule forms the J-aggregates at the surface and acts as either energy donor or acceptor. The number of monomer

units comprising the J-aggregatearranged in brick-stone structure12is about 20. Excitation energy transfer is possible only when the distance between the donor aggregate and the acceptor aggregate is smaller than Ro. luminescence properties of the liposomes incorporating several combinations of the J-aggregate of cyanine dyes given in Figure 1. Dye-IV molecules were adsorbed a t the liposomal surface giving rise to the J-aggregate. In the absence of the DMPC:DCP liposomes, the dye-IV solution shows a M-band (A = 590 nm) and a D-band (A = 542 nm), together with weak monomer fluorescence (A = 625 nm). As shown in Figure 7A, an intense J-band (A = 686 nm, e = 1.38 X lo5 M-' cm-l) together with resonance fluorescence (A = 702 nm) appeared after adding the liposomes. The intensity of resonance fluorescence was weaker than those of the dye-IIand the dye-I11aggregates. The fitting parameters in eq 1were B = 1.54 and C = 0.63. Absorption and fluorescence spectra of the dye-1I:dyeIV liposome are shown in Figure 7B. Quenching of resonance fluorescence of the dye-I1 aggregate (A = 608 nm) and sensitization of that of the dye-IV aggregate (A = 702 nm) are evident. Judging from the absorption spectra, dye11 and dye-IV form the J-aggregate independently at the liposomal surface. After the correction of the reabsorption of the dye-IV aggregate, ln(H1) and ln(1 + H2) were plotted versus r(dye-IV) by use of q of 1.1. As was the case of the dye1I:dye-I11liposomes, both plots were represented by two straight lines due to the influence of 2. After the correction of quenching and sensitization coefficients according to eqs 13 and 15 with Xbt = 1.3,both plots could be fitted to the single straight line passing through the original point (Figure 5B). The slope of 2.8-3.0 nm2 is nearly equal to that of the dye-1I:dye-I11 liposomes. A fluorescence peak at A = 650 nm as seen in Figure 7B is intense at low r(dye-IV), and became weaker with increasing r(dye-IV). It may be possible that this fluorescence comes from 2 in the dye-1I:dye-IV liposomes. We next examined excitation energy transfer from the dye-I aggregate at the liposomal surface. In this case, was 430 nm. A M-band (A = excitation wavelength, b,,, 522 nm) and a D-band (A = 488 nm) in the dye-I solution

3400 Langmuir, Vol. 9, No. 12, 1993

Sato et al. cs

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Wavelength ( n m ) Figure 7. Absorption (solidlies) and fluorescence (brokenlines) spectra of dye-IV solution in the presence or absence of the DMPC:DCP = 1:l liposomes (A) and dye-IIdye-IV liposome (B). Excitation wavelength &= = 450 nm. The scale of fluorescence intensityis the same as that in Figure 2. (A) a, 0.33 vol % CHsOH/H20 solution of dye-IV (1.0 X 106 M); b, c, 0.31 vol 5% CHsOH/H20solution of dye-IV (9.4 X 1o-BM) and liposome ([DMPC + DCPI = 9.1 X 106 M). (B)a, b, 0.6 vol % CHaOH/ H20 solution of dye-I1 (9.4 X 1o-B M), dye-IV (9.4 X 1o-BM), and DMPC:DCP = 1:l liposome ([DMPC + DCP] = 9.1 X 106 M).

decreased, and a narrow J-band (A = 580 nm, t 1 1 X lo6 M-1 cm-1) with resonance fluorescence (A = 584 nm) appeared after the addition of the DMPC:DCP liposomes. As shown in Figure 8A(c), resonance fluorescence of the dye-I aggregate is more intense than those of the dye-11, dye-111, and dye-IV aggregates. Absorption and fluorescence spectra of the dye-1:dyeI11 liposome and the dye-Idye-IV liposome are shown in Figure 8. Quenching of resonance fluorescenceof the dye-I aggregate and sensitization of the dye-I11 or the dye-IV aggregates are observed. After the correction of reabsorption as well as the influence of 2,quenching and sensitization plots for the dye-1:dyeIV liposomes with parameters 9 = 0.077 and Xht = 1.3 are made to fit to single straight line passing through the original point. The plots for the dye-1:dye-I11liposomes with parameters 9 = 0.45 and Xht = 2.0 somewhat deviated from the straight line. The slopes of the quenching and sensitization plots, i.e., the area of the "Perrin volume" for the all donor-acceptor combinations in this study are summarized in Table I. The area of the "Perrin volume" is related to the fluorescence lifetime of the donor and the rate constant of energy transfer, as well as the size of the J-aggregate. Therefore, the rate constant of energy transfer from the dye-I1 aggregate to the dye-I11 aggregate and that to the dye-IV aggregate would be nearly equal to one another, if the number of monomer units consisting of a J-aggregate is almost the same. When dye-I aggregate is the donor,

Figure 8. Absorption (solidlines) and fluorescence(brokenlies) spectra of dye-I liposome and dye-Idye-I11liposome (A) and dye-Idye-IVliposome (B).Excitation wavelength &x = 430 nm. The scale of fluorescence intensity is same as that in Figure 2. (A) a, c, aqueous solution of dye-I (1.0 X 1V M) and DMPC: DCP = 1:l liposome ([DMPC + DCPI = 9.1 X 106 M); b, d, aqueous solution of dye-I (8.6 X 1o-B M), dye-I11(8.6 X 1o-B M), and DMPC:DCP = 1:l liposome ([DMPC + DCP] = 8.3 X 106 M); (B)a, b, 0.31 vol % CHsOH/H20 solutionof dye-I (9.4 X 1o-B M), dye-IV (9.4 X 1o-B M) and DMPC:DCP = 1:l liposome ([DMPC + DCP] = 9.1 X 1W M). Table I. Slopes of the Perrin-Type Plot for the Combinations of Two Kinds of J-Aggregates. acceptor dye-I11 dye-IV donor (A, = 660 nm) (A, = 690 nm) dye-I (A, = 580 nm) 0.8-1.0 nm2 1.0-1.2 nm2 dye-I1 (A, = 600 nm) 2.8 nm2 2.8-3.0 nm2 A,

is the wavelength at the J-band.

the slope is smaller than the dye-I1aggregate. One reason would be the different fluorescence lifetime between dye-I and dye-I1 aggregates. However, the behavior of dye-I aggregate is rather complicated. The intrinsic trap state of the dye-I aggregateSomay influence energy transfer process. Further studies are needed to clarify energy transfer from dye-I aggregate, as well as the detail characterization of 2. 5. Conclusions

Liposomes incorporating two kinds of cyanine dyes were prepared. Excitation energy transfer between the J-aggregates was really observed in the combinations dye-I dye-111, dye-1:dye-IV,dye-IIdye-111,and dye-IIdye-IV at the liposomal surface. These dye molecules are arranged in the brick-stone structure on the liposomes, forming the donor aggregate and acceptor aggregate independently. It has been shown that J-aggregates distribute randomly but the distance between them is close enough for energy (30)Muenter,A.A.;Brumbaugh,D.V.;Apolito, J.;Hom,L.A.;Spano,

F. C.;Mukamel, S.J.Phys. Chem. 1992, 96,2783.

Luminescence Properties of Liposomes transfer to occur. The number of monomer units consisting of one J-aggregate is estimated to be about 20. In the present study, energy transfer efficiency has been analyzed by the crude Perrin type kinetics. Energy transfer between the J-aggregatesoccurred when the donor and acceptor aggregates are located within the distance less than the physical size of the J-aggregate. In this situation, the conventional Forster theory is not directly applicable. An examination of the fluorescence decay k i n e t i ~ P - 3of~ our systems is necessary to get further

Langmuir, Vol. 9, No. 12, 1993 3401 insight to the geometry of the J-aggregates and the mechanism of energy transfer between the J-aggregates.

Acknowledgment. We thank Dr. T. Tanaka and Dr. Y. Wada of the Department of Chemistry and Materials Technology, Kyoto Institute of Technology, for allowing us to use the spectrofluorometer MPF-4 and the spectrophotometer 2W-20. (31)Tsubomura, T.;Sakurai, 0.; Morita, M. J. Lumin. 1990,45,263.