Pergamon Press

Life Sciences Vol . 19, pp . 1743-1750, 1976 . Printed in the U .S .A .

IMPROVED

ENTRAPMENT OF DRUGS

Kaoru Tsujii *,

Junzo

I~d MODIFIED LIPOSOMES

Sunamoto ** and

Janos

H.

Fendler

Department of Chemistry, Texas A&M University 77843 College Station, Texas (Received in final form October 22, 1976) Summary Incorporation of 8-azaguanine and 6-mercaptopurine into single compartment dipalmitoyl-DL-a-phosphatidylcholine liposomes has been increased dramatically by the presence of chloranil transfer complex formation . The enhanced entrapment is due to charge . The charge transfer complex readily decomposes to the parent donor drug and chloranil acceptor . Chloranil, however, may be toxic . Using 3,5-dinitrobenzoyl-n-butylamide and 3,5-dinitrobenzoyl phosphatidylethanolamine as electron donors did not result in enhanced entrapments . Single and multicompartment liposomes (1-3) are increasingly being utilized as immunological adjuvants, carriers for enzymes The proposed delivery mechanism involves the and drugs (4-16) . intact entry of the liposome encapsulated drug into the cell, conceivably by endocytosis, where lysosomal lipases or other The attractiveness of liposome mediated factors release it (8) . drug delivery is that toxicological and immunological effects are minimized and that the carrier is biodegradable . We have recently demonstrated the incorporation of 8-azaguanine, an antimetabolite, into positively charged single and multiple component dipalmitoylDL-a-phosphatidylcholine and egg yolk phosphatidylcholine liposomes (18) . Although the liposome encapsulated 8-azaguanine was shown to be nontoxic, only a marginal increase in the survival rate of Leukemia L-1210 bearing mice was observed . This somewhat disappointing result was due, at least in part, to the inefficient entrapment of the drug . The extent of encapsujation of 8-azaguanine in single compartment liposomes is only 0 .70-1 .80% (18) . It is felt, therefore, that optimization of drug entrapment and release are of paramount importance in the development of functional liposomes which are able to affect selective delivery to given receptors . The present work reports dramatic enhancements of drug encapsulation by charge transfer complexation . The role of charge transfer interactions have been recognized in biolog (19-24) and purines have been shown to be electron donors (19,20 . $-azaguani.ne, and 6-mercaptopurine were chosen as donors, while chloranil, 3,5-dinitrobenzoyl-n-butyl-amine and 3,5-dinitrobenzoyl phosphatidylethanolami.d e have been utilized as acceptors . On leave from Kao Soap Company, Tokyo, Japan . On leave from the Department of Industrial Chemistry, Nagasaki University, Nagasaki, Japan . 1743

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Drugs in Modified Liposomes

Vol . 19, No . 11

Materials and Methods Synthetic dipalmitoyl-DL-a-phosphatidylcholine (Sigma, Grade I), 8-azaguanine (Calbiochem) and 6-mercaptopurine (Nutritional Biochemicals Co .) were used without further purification . 8azaguanine-2-'"C and 6-mercaptopurine-8-'"C were purchased from New England Nuclear and ICN Pharmaceuticals, Inc ., respectively . Cholesterol (MCB Co .) and chloranil (MCB Co .) were recrystallized 3,5-dinitrotwice from ethanol and chloroform respectively . benzoyl-n-butylamide (DNB-BA) and 3,5-dinitrobenzoyl phosphatid lethanolamide (DNB-PE) were synthesized by standard procedures (25~ . Double distilled water was used in all experiments . Chloroform stock solutions of dipalmitoyl-DL-a-phosphai;i.dylcholine, cholesterol, chloranil, DNB-BA and DNB-PE were stored Stock solutions in a refrigerator for given sets of experiments . were freshly prepared, however, every 2 weeks . Appropriate amounts of the phospholipid, cholesterol and acceptor stock solutions were mixed (phosphatidylcholine : cholesterol : acceptor = 16 .3 : 10 .3 : 2 .0 ; in mole ratios) in a round bottom flask and the solvent was evaporated to dryness in a rotary evaporator under reduced pressure . Complete removal of the organic solvent was accomplished by storing the round bottom flask in a desiccator The remaining thin film was dispersed by overnight under vacuum . adding 2 .0 ml of aqueous solution of the 1 "C labelled (0 .25 uC/2ml) donor (. 8-azaguanine or 6-mercaptopurine) containing O .lOM NaCI 5 .5mM buffer (sodium acetate for pH = 6 .0, potassium phospate for pH = 7 .0 and 8 .0 and sodium borate for pH = 9 .0 and 10 .0) . The final concentrations of the phospholipid and cholesterol were Dispersion was carried out at 508 .2mM and 5 .2mM, respectively . 60° using the microprobe of a Braunsonic 1510 sonifier at 70 watts The clear sonicated liposome solution was passed for 15 minutes . through a Sephadex G-50 column to separate the free drug from that entrapped in the liposome . Radioactivity was determined by means of a Beckman LS-.100 liquid scintillation counter . The counting cocktail was prepared by dissolving 60 g of naphthalene, 4 g of PPO, 0 .2 g of POPOP and 20 ml ethylene glycol in 1 .0 liter dioxane . The activity of the free or liposome encapsulated drug was determined in each fraction by blending 1 .0 ml solutions into 10 ml of the cocktail . Percentages of incorporation were calculated by relating the specific activities under the peaks due to the lipoRecoveries of some encapsulated drug to the total activity . radioactivity were generally above 95~ . Pre-coated silica gel 60F254 TLC sheets (Brinkman Instruments Inc .) were used for thin layer chromatography . The eluent was nChloranil, 8-azaguanine and 6 butanol ; water (84 ; 14, v/v) . The phosmercaptopurine were detected by an ultraviolet lamps . pholipid has visuali.zed by iodine, Absorption spectra were determined on a Cary 118C spectrophotometer at ambient temperature using a pair of matched 2 .0 cells, Spectra were obtained within 10 minutes subsequent to mixing, Absor tion spectra of liposome solutions were determined on ultracentri~uged (using a Beckman L-2 ultracentrifuge, rotor type 65B, 45,000 rpm, 60 minutes) solutions . pH were determined by means of a Radiometer PHM 26 pH meter . Results and Discussion Percentages of the incorporation of 6-mercaptopurine and 8-

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Drugs in Modified Liposomea

174 5

azaguanine in single compartment dipalmitoyl-DL-a-phosphatidylchoAssuming that external line liposomes are given in Table I . diameter of single compartment liposomes is approximately 250 A,

Table

I .

Incorporation of 6-Mercaptopurine-8-'"C and 8-Azaguanine-2- 1 "C in Liposomes a

Acceptor

None

Chlorani.l

WB-BA

DNB-PE

% Incorporationb

Drug H = 7 .0

6-Mercaptopurine

0 .45(1 .7)

,18(0 .66) 0 .10(0 .37) 0 .24(0 .88) 0 .44(1 .6)

8-Azaguanine

0 .86(3 .2)

6-Mercaptopurine

26 .7(98)

8-Azaguanine

5 .7(21)

6-Mercaptopurine

0 .24(0 .88)

8-Azaguanine

0 .89(3 .3)

6-Mercaptopurine

0 .32(1 .2)

2 .2,28 .9 (118,106)

pH = 8 .0

14 .6(54)

.9(18)

pH = 9 .0

pH = 10 .0

pH = 6 .0

0 .76(2 .8)

0 .90(3 .3)

4 .9(18)

5 .6(21)

4 .0(15)

1 .8(6 .6)

0 .08(0 .29) 0 .21(0 .77) 0 .87(3 .2) 0 .81(3 .0)

0 .97(3 .6)

0 .19(0 .70)

a Single compartment liposomes prepared from s nthetic dipalmitoyl-DL-aphosphatidylcholine (12 mg), cholesterol (4 mg~ . Each preparation contained 1 mfA drug and 1 mM acceptor . b

Values in partenthesis are the number of drug molecules per single compartment liposane . and that each li.posome contains 3000 phospholipid molecules (26,27) the number of vesicles per umole of phospholipid was calculated to be 2x10'" (28), Using this value allowed the calculation of the number of drug molecules per liposome . These values are also given in Table I . DNB-BA and DNB-PE do not appreciably enhance tüe i.ncorpgrati,on of 6-mercaptopurine or 8-azaguanine . Chloranil exerts, however, a dramatic effect . At pH = 7 .0 entrapment of 6mercaptopurine is enhanced from 0 .18% to 31% . Similarly, in the presence of chjoranij at pH = 6 .0 5 .7% 8-azaguanine is encapsuled but i.n its absence the uptake i.s only 0 .86%, Extent of drug entrapment is markedly pH dependent (Table I) . The enhanced drug uptake into liposomes are rationajized in terms of favorable charge transfer complex formation .

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Druga in Modified Liposomea

Figure 1 illustrates the complexed 6-mercaptopurine in

Vol . 19, No . 11

absorption spectra of free and aqueous buffer solutions at pH = FIG .

6 .0 .

1

Absorption spectra of 6-mercaptopurine, chloranil and the mixture of them in pH = 6 .0 buffer solution ; -s 5x10 M 6-mercaptopurine ( ), 5x10 s t4 chloranil (-+-+), -s s 5x10 M 6-mercaptopurine + 1x10 M chloranil (---), and 5x10 s M 6-mercaptopurine + 5x10 -s M chloranil (- .- .) . The insert shows a plot of the data according to equation 1 . Absorbantes were determined at 410 nm .

Charge transfer complex formation is clearly evident . Indeed it is possßble to calculate the association constant for the intepaction between 6-mercaptopurine and chloranil by using the modified Benesi-Hildebrand equation (29) 1 : 1 ~ E -

K CD

1 (EA D -

EA )

~

1 [D]o

+

1 E AD -

EA

[1]

where E~ and E AD are mo~dr absorptivity of the acceptor and the complex respectively, K the association constant and [D], is the total concentration c ofa the donor An apparent molar absorptivity of the acceptor, E = A'/[A], where A' and [A], are the optical densities at wavelength a and the concentration of the acceptor, respectively . From the A Bbserve~ D linear relationship (Insert in Figure 1) values f_or K and E wer calculated to be 2x10 3 M ' and 2 .6x10 3 M - ' cm ' . Tote value a for K~ D indicates strong association . Charge transfer association between 6-merca topurine and chloranil is also evident in liposomes (Figure 2~ . These charge transfer complexes are not very stable, however . They cannot be Isolated under our experimental conditions or be detected by thin-layer chromatography (Table II) . Their decomposition may, i. n fact, be facilitated under the conditions of chrômatography . 6-Mercaptopurine has an R value of 0 .66 . The obtained similar R -values on spotting the ~-mercaptopurine-chloranil charged t~ansfer complexes in aqueous buffer (R G = 0 .64) or in

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174 7

Drugs in Modified Liposomes

liposomes (R = 0 .66) substantiates this postulate . The 8-azaguanine-chloranil charge transfer complex behaves analogously (R values for spotting 8-azaguanine in ethanol in the form of its chloranil complex in ethanol and in the liposome are 0 .61, 0 .61, and 0 .59, respectively) .

FIG .

2

Absorption spectra of chloranil and the mixture of -s M chloranil and 6-mercaptopurine in liposomes ; 3 .2x10 s M 6-merpcaptopurine chloranil (-+-+-), chloranil + 6 .5x10 -s M 6-mercaptopurine ( ) and (- .- .-), chloranil +_ 9 .5x10 chloranil + 13 .0x10 s M 6-mercaptopûrine (-----) . The reference cell contained identical liposome solution but without any chloranil or 6-mercaptopurine .

TABLE II . R F Values a Sample

RF

6-Mercaptopurine in aqueous buffer at pH = 6 .0 6-Mercaptopurine in ethanol 8-Azaguanine in ethanol Chloranil in ethanol Chloranil in chloroform Lipbsome in ethanol Liposame in chloroform 6-Mercaptopurine + chloranil (1 :1) in aqueous buffer^ at pH = 6 .0 8-Azaguanine + chloranil (1 :1) in ethanol 6-Mercaptopurine + chloranil in liposome 8-Azaguanine + chloranil in liposome a 0n thin-layer plates .

0 .61

0 .66 0 .66

0 .06 0 .06

0 .83 0 .83

0 .64 0 .83 0 .83 0 .08 0 .66 0,82 . 0 .07 0 .59 0 .84 0 .61

See Material and Methods for details .

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Druga in Modified Liposomea

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The present work demonstrated that in suitable cases charge transfer complex formation can substantially enhance drug incorSubsequent to their uptake, these comporation into liposome . Efforts are plexes decompose to their donor and acceptor parents . underway to form charge transfer complexes which do not have harmful pharmacological effects and to test their deliveries _in vivo . Acknowled gments Support of this work by the National Science Foundation, the United States Energy Research and Development Administration and the Robert A . Welch Foundation is gratefully acknowledged . References 1, 2. 3. 4. 5, 6. 7. 8, 9, 10, 11, 12, 13, 14 . 15, 16, 17 . 18, 19 . 20, 21 . 22, 23,

A . D . BANGHAM~ in Pro ress in Bio h sics and Molecular Biology (J . A . V, GU LER and L , Eds , _18, 29-95 Pergamon Press, Oxford (1968) . A, D, BANGHAM, M . W . HILL, and N . G . A . MILLER, in t4ethods in Membrane 6iolo : (E . D . Korn, Ed), _11, (P . 38) Plenum~ress, New orc 1974 , D . PAPÂHADJOPOULOS and K, K, KIMELBERG . Prog . Surface Sci . , 4, 141 (1973), G. GREGORIADIS, FEES Lett ., _36, 292-296 (1973) . Y . E . RAHMAN, E, , C RN , S L . TOLLAKSEN, B . J . WRIGHT, S . L, NANCE and J,F . THOMSON, Proc, Expt, Biology and Medi.ci.ne, 146, 1173-1176(1974) . ~~RIA~6TS and A, C . ALLISON, FEBS Lett ., 45, 71-74 (1974) . C, W, M, GRANT and H, M . MCCONNEL, roc, atl .~Ccad * Scie . U, S  71, 4653-4657 (1974) . ~6REG~IADIS, C .P . SWAIN, E . J . WILLIS AND A . S . TAVILL, The Lancet, 1313-1316 (1974) . . C, A SON and G, GREGORIADIS, Nature, 252, 252 (1974) . G . GREGORIADIS, D, PUTMAN, L . LOUIS - an~D . NEERUNJUN, Biochim J , 140, 323-330 (1974) . C OTiIand S, C, KINSKY, Biochemistry , 14, 23312337~(1975), C, R . ALVING, D . H . CONRAD, J, P, GOCKERMAN, M . B, GIBBS and G, H, WIRTZ, Biochim, Bio h s Acta, 394 157-165 (_1975), R . L, JULIANO and , S , oc em, i.op~ys, Res, Commun, 63, 651-658 (1975), +I, R, REDWOOD, V, K, DANSONS and B, C . PATEL, Biochim, ~Bio~~hy,~s ~Acta, 406, 347-361 (1975), A . W, SEGAL, G, GREGORIADIS a. nd C, D . V, BLACK, Clinical Science and Molecul r Medicine, 49, 96-106 (1975~~ J, DR N K and L, AUGENSTEIN, Photochemistry and Photobio 0 5, 83x97 (1966) ; L, W, LAW, Cancer esearch 1 , 61 0) . M . COLLEY AND B, E, RYMAN, B~Qchem, oc . ~ansactions , _3, 157-159 (1975), J . H . FENDLER and A . ROMERO, Life Sciences, 18, 1453 (1976) . P, MACHMER and J . DUCHESNE, Nature, 1 18-619 (1965), M . A, SLIFKIN, Biochim . Bio Fs~Ccta,103, 365-373 (1965) . J, B . BIRKS and M . , SLIFKI , ature , 1~, 42-45 (1963) . M * A . SLIFKIN, S ect ochim, Acta, 20, 1~-1554 (1964) . R, FOSTER and P, H N oc m . Biophys . Acta , _112, 482-489 (1966),

lr.

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24 . 25 . 26 . 27 . 28 . 29 .

Druge in Modified Liposomes

1749

R . FOSTER and C . A . FYFE, Biochim . Biophys . Acta , 112, 490-495 (1966) . R . B . WAGNER and H . D . ZOOK, S nthetic Or anic Chemistr , John Wiley and Sons, Inc ., New Yor , 1 53, P . S . M . JOHNSON, A . D . BANGHAM, M . W . HILL and E . D . KORN, Biochim . Bio h s . Acta, 233, 820-526 (1971) . C . H . HU N , Biochemistr,8, 344-352 (1969) . D . PAPAHADJOP SIÉ and E . MAYHEW, Biochim . Bio h s . Acta, 363 404-518 (19?~) . R. r anic Char e-Transfer Com lexes, Academic Press, , p . 13 . New York (1