[CANCER RESEARCH 45, 128-134, January 1985]

Activation of Alveolar Macrophage Tumoricidal Activity and Eradication ofExperimental Métastasesby Freeze-Dried Liposomes Containing a NewLipophilic Muramyl Dipeptide Derivative1

Nigel C. Phillips,2 Maria L. Moras, Louis Chedid, Pierre Lefrancier, and Jean M. Bernard

ImmunothérapieExpérimentale.Institut Pasteur, 28, rue du Dr. Roux, 75724 Paris Cedex 15 [N. C. P., M. L. M., L C.], and Institut Choay, 10, rue Morel, 92120 Montrouge[P. L, J. M. B.], France

ABSTRACT

The ability of a member of a new class of lipophilic muramyldipeptide (MDP) derivative, muramyl dipeptide-glyceryldipalmi-tate (MDP-GDP), to induce alveolar macrophage cytotoxic activ

ity in vitro towards B16 melanoma cells when incorporated intotwo types of liposome was studied. MDP-GDP incorporated intoconventionally prepared liposomes formulated from distearoyl-

phosphatidylcholine and phosphatidylserine (7:3 molar ratio) was10-fold more effective than liposomes containing MDP, and7000-fold more effective than free MDP in inducing macrophagecytotoxic activity. MDP-GDP incorporated into freeze-dried liposomes was 50,000- to 100,000-fold more effective than freeMDP in inducing such activity. Freeze-dried liposomes containingMDP-GDP were efficiently localized in the lungs of normal mice,

and induced cytotoxic activity in the alveolar macrophages. Suchliposomes were able to significantly reduce the pulmonary met-

astatic burden of mice carrying the B16 melanoma. These dataprovide evidence that this class of lipophilic MDP derivative,when incorporated into freeze-dried liposomes, is a potent in-

ducer of macrophage cytotoxic activity in vitro and in situ, andhas antitumor activity in vivo. In addition, the use of a freeze-drying procedure allows the preparation and long-term storage

of reproducible liposome formulations.

tumoricidal activity compared to free MDP (28, 31). Such preparations have been shown to activate alveolar macrophages invivo with a concomitant eradication of established pulmonarymétastases.The use of hydrosoluble MDP for liposomal entrapment poses problems in terms of efficient incorporation (10) and,because of its low molecular weight, subsequent retention (24).Attention has therefore been focused on lipophilic MDP derivatives which are capable of insertion into the liposome membrane.The ability of 2 such derivatives, MTP-PE, where MTP is conjugated to phosphatidylethanolamine, and 6-O-stearoyl MDP,

where MDP is conjugated to stearic acid, to stimulate macrophage cytotoxic activity has been recently described (12, 29,31). However, the method of preparation of liposomes containingsuch derivatives, vortexing of dried thin lipid films, presents anumber of problems in terms of time, reproducibility, and long-

term storage.In this paper, we demonstrate that a member of a new class

of lipophilic MDP derivative, MDP-GDP, is a potent inducer of

macrophage cytotoxic activity in vitro when incorporated intoliposomes. Such liposomes are capable of activating alveolarmacrophages in situ and can reduce the pulmonary metastaticburden of mice carrying the B16 melanoma. The use of a freeze-

dried formulation is described which enables identical liposomebatches to be prepared and kept under long-term storage.

INTRODUCTION

MDP3 is the minimum structure possessing immunoadjuvantactivity which can be isolated from and replace whole mycobac-teria or Freund's complete adjuvant (3, 13). Since the original

structure was elucidated, numerous analogues and derivativeshave been synthesized and evaluated for biological activity (1).In addition to its ability to stimulate a number of macrophageactivities such as monokine production (4, 9, 14), collagenaseand prostaglandin secretion (33), and Superoxide aniónsecretion(16), MDP is capable of indirectly inhibiting tumor cell growth invivo (20) and of inducing macrophage-mediated tumoricidal ac

tivity in vitro (32).The use of liposomes to deliver MDP to macrophages in vitro

has been shown to result in a considerable enhancement of

'The support of l'Association Claude Bernard, 75004 Paris, France (N. C.

Phillips) is gratefully acknowledged.2To whom requests for reprints should be addressed.3The abbreviations used are: MDP, N-acetylmuramyl-L-alanyl-o-isoglutamine;

MDP-GDP, «-(N-acetylmuramyl-L-alanyl-o-isoglutaminyl),#,7<)ipalmitoyl-s/i-o,lyc-

erol; DSPC, distearoylphosphatidylcholine; PS, phosphatidylserine; PBS, phosphate-buffered saline; MEM, minimal essential medium supplemented with Earles'salts; PCS, 5% heat-inactivated fetal calf serum; [125IJIdUrd, [125l]-5-iododeoxy-

uridine; MTP-PE, muramyl tripeptide conjugated to phosphatidylethanolamine;[14C]DSPC, di[l-14C]stearoylphosphatidylcholine.

Received March 7, 1984; accepted September 10, 1984.

MATERIALS ANO METHODS

Animals. Male C57BL/6J mice, 6 to 8 weeks old, and male F344 rats,200 to 220 g, were obtained from Iffa Credo (L'Arbresle, France). All

animals were specific pathogen free and were maintained on chlorine-

free drinking water for 2 weeks prior to use.Tumor Cell Line. The murine melanoma line B16-BL6, syngeneic for

C57BL/6 mice, was obtained from Dr. A. Bogden (E. G. and G. MasonResearch Institute, Worcester, MA), and maintained in monolayer culture.

Culture Media. All cultures were carried out at 37° in a humid

atmosphere containing 5% CO: using MEM supplemented with 1 ITIMglutamine, 1 ITIMsodium pyruvate, vitamins, nonessential amino acids,streptomycin (100 Mg/ml), penicillin (100 units/ml) (all from Flow Laboratories S. A., Asnieres, France), and 5% heat-inactivated PCS (Gibco

Europe, Uxbridge. Middlesex, United Kingdom).Reagents. MDP was MDP-GIRPI (Choay Chimie Réactifs,Paris,

France). [6-3H2]-A/-acetylmuramyl-L-alanyl-D-isoglutamine (specific activ

ity, 2 Ci/mmol), was synthesized by Institut Choay. The detailed synthesisof MDP-GDP will be reported elsewhere. Briefly, fert-butyloxycarbonyl-L-alanyl-D-isoglutamine was incubated with /S.-y-dipalmitoyl-sn-glycerol inthe presence of benzotriazol-1-yloxy-tris(dimethylamin)-phosphoniumhexafluorophosphate, N-methylmorpholine, and imidazole for 48 hr at

ambient temperature in tetrahydrofuran:dimethylformamide (1:1). Theresultant product was extracted with chloroform, and placed on a silicagel column equilibrated with chloroform; «-{tert-butyloxycarbonyl-L-alanyl-D-isoglutaminyl), /3,7-sn-glycerol was obtained by successive elu-

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tion with chloroform, chloroform:methanol (50:1), and chloro-

fomr.methanol (25:1). After ultrafiltration, the product was lyophilizedfrom its dioxane solution. The product was A/-deprotected by treatment

with normal hydrochloric acid in glacial acetic acid, and isolated bylyophilization from its acetic acid solution. The ensuing product, <*-(L-alanyl-o-isoglutaminyl), /J.-y-dipalmitoyl-sn-glycerol, was incubated with«-benzyl-4,6-)-O-benzylidene-A/-acetylmuramic acid in the presence ofbenzotriazol-1 -yloxy-tris(dimethylamin)phosphonium hexafluorophos-phate and A/-methylmorpholine for 36 hr in chlorofornrdimethylformamide

(5:2). The product was extracted with chloroform and placed on a columnof silica gel equilibrated with chloroform:methanol (15:1); «-[/V-acetyl-muramyl-(a-benzyl-4,6-O-benzylidene)-L-alanyl-D-isoglutaminyl],/3,7-di-palmitoyl-sn-glycerol was placed in glacial acetic acid, and hydrogenated

in the presence of 5% palladium charcoal for 40 hr. After removal of thecatalyst, MDP-GDP was obtained as a lyophilized powder from its aceticacid solution ([«]?5:+ 23.4°;c, 0.7, acetic acid).

,5, 1.6% CH3COOH

Calculated:C 60.3,H 9.2N 4.9

Found: C 60.3,H 9.2N 5.0

Liposome Preparation. DSPC was purchased from Sigma ChemicalCo. (St. Louis, MO). PS was purchased from Serva Feinbiochemica(Heidelberg, Federal Republic of Germany). [14C]DSPC (specific activity,

115 mCi/mmol) was purchased from Amersham International (Amer-sham, Buckinghamshire, United Kingdom). All phospholipids were chro-matographically pure and were stored at -40° under N2. To prepare

multilamellar liposomes, DSPC and PS (7:3, 7:0.3, or 7:0 molar ratios)were admixed with MDP-GDP in anhydrous chloroform, and rotaryevaporated to dryness in round-bottomed glass flasks. The dried lipidfilm was rehydrated with PBS at 20°for 10 min, and then vortexed at60°to form liposomes. Control liposomes were prepared by omitting the

MDP-GDP. Hydrosoluble MDP was incorporated into liposomes by adding PBS containing MDP (0.05 to 5 mg/ml), spiked with W-acetylmuramyl-L-[6-3H2]alanyl-D-isoglutamine to dried films of DSPC:PS (10 ^mol phos-

pholipid, 7:3 M ratio). The liposomes were subsequently washed 3 timesin 20 ml of PBS by centrifugation at 10,000 x g at 4°for 30 min. The

percentage of incorporation of MDP was determined by liquid scintillationcounting of the starting MDP solution and of the final liposome preparation. The recovery of phospholipid was determined by adding a spike of[14C]DSPC. All counts were quench-corrected before calculation of in

corporation or recovery. Freeze-dried liposomes were prepared by dissolving DSPC, PS, and MDP-GDP in anhydrous 2-methyl-2-propanol at28-30° and placing aliquots containing the required quantities in 5 ml of

freeze-drying vials (total volume, 1 ml). After freezing the 2-methyl-2-propanol solutions at 4°,the vials were subjected to lyophilization for 6hr at 20°.Freeze-dried lipids were stored at -20° or +4° until required.To prepare liposomes, the required volume of PBS at 60°was added to

the lipids, which were then vortexed for 2 min and stored on ice for 30min before use.

All preparations were carried out under sterile conditions using apy-

rogenic materials.Liposomal Incorporation and Retention of MDP. Liposomes were

prepared from 10 ¿¿molof total phospholipid (DSPC:PS; 7:3 M ratio) and5 ml of a 5-mg/ml solution of MDP containing a spike of [3H]MDP. After

the removal of nonincorporated MDP by 3 cycles of centrifugation at10,000 x g, the liposome preparation was placed in 2.0 ml of MEM orMEM-FCS and incubated at 37°in a humid atmosphere containing 5%

CO2. At timed intervals, triplicate aliquots (100 fi\) were removed, dilutedto 1.0 ml with MEM or MEM-FCS, and centrifugea at 10,000 x g for 30min at 4°.The resultant liposome pellets were resuspended in 500 n\ of

PBS, and the radioactivity determined by liquid scintillation counting. The

incorporation and retention of MDP was corrected for the loss of smallliposomes during the centrifugation procedure by reference to duplicateliposome preparations labeled with [14C]DSPC. No significant radiolysisof [6-3H2]-W-acetylmuramyl-L-alanyl-D-isoglutamine was found to occur

under these experimental conditions.Macrophage-mediated Cytotoxic Assay. The relative efficacy of free

MDP, liposome-encapsulated MDP, or liposome-incorporated MDP-GDPin inducing alveolar macrophage-mediated cytotoxicity in vitro was de

termined using a modified version of the radioactive release assaydescribed by Sonéand Fidler (28). Because of the large number of cellsrequired in these studies, F344 rats were used as the source of alveolarmacrophages, and the effect of liposome treatment on their cytotoxicactivity was determined using xenogeneic murine B16-BL6 melanoma

cells. Previous reports have shown that the in vitro treatment of suchmacrophages with liposomes containing MDP or macrophage activatingfactor results in the induction of comparable cytotoxic activity towardssyngeneic, allogeneic, or xenogeneic tumor target cells, but not towardsnontumorigenic cell lines (27, 28, 30). Alveolar macrophages were isolated from F344 rats which were anesthetized with 0.5 ml of Nembutali.p. (5 mg/100 ml; Laboratoires Abbott S.A., St.-Rémy-sur-Avre, France),

and exsanguinated by cannulation of a renal artery. The trachea wasthen exposed and cannulated, and the lungs of each rat were washedwith 9 x 5 ml of PBS at 37°.The washings were pooled and centrifugedat 800 x g for 10 min at 4°,and the resultant cell pellet was resuspended

in MEM-FCS. After a further washing by centrifugation, the cells wereresuspended in MEM-FCS to give a macrophage concentration of 5 x105/ml. The cell population was determined to be >95% macrophage by

morphology and naphthyl esterase staining. Aliquots (100 *J) were thenplaced in the wells of microtiter plates (Titerteck, 76-002-05; surface

area, 0.28 sq cm). After incubation for 4 hr, nonadherent cells (<5%)were removed by extensive washing with MEM-FCS. The macrophage

monolayers were then incubated with the liposome preparations or withhydrosoluble MDP in a final volume of 200 /<lfor 24 hr. The macrophagemonolayers were then washed 3 times with 200 n\ of MEM-FCS, afterwhich 100 n\ MEM-FCS were added to each well. B16-BL6 target cellsin exponential growth phase were incubated with [125l]ldUrd (0.2 pCi/ml;

specific activity, 2200 Ci/mmol; New England Nuclear, Boston, MA) for24 hr. The cell monolayers were washed extensively with warm serum-free MEM to remove unincorporated [125l]ldUrd, after which the cells

were detached by a brief (1 min) exposure to 0.25% trypsin:0.02% EDTAin PBS and were suspended in MEM-FCS. After being washed bycentrifugation at 800 x g for 10 min at 4°,the cells were resuspendedin MEM-FCS to give a viable cell concentration of 5 x 104/ml. Aliquots

(100 n\) were added to the pretreated macrophage monolayers, andcoculture carried out for 72 or 96 hr. The plating efficiency of the B16-

BL6 cells was determined to be 90 to 95%. At the end of the incubationperiod, each well was washed 3 times with 200 n\ of warm PBS, and theremaining adherent cells were lysed by the addition of 200 ¡Aof 0.5 MNaOH. The lysate and 2 washes (2 x 200 ¡Aof 0.5 M NaOH) from eachwell were combined and counted for radioactivity. All determinationswere carried out in triplicate. The percentage of specific cytotoxicity wascalculated using the following:

(Mean cpm target cells + control macrophages)- (mean cpm target cells + treated macrophages)

(Mean cpm target cells+ control macrophages)

The effect of treatment of macrophage-mediated cytotoxic activity wasanalyzed statistically using Student's 2-tailed t test.

In Vivo Distribution of Liposomes Containing MDP-GDP. Freeze-

dried liposomes were prepared using DSPC:PS (7:3, 7:0.3, or 7:0 Mratios) spiked with [14C]DSPC and containing 10 ng of MDP-GDP per

0.5 ¿imolof phospholipid. C57BL/6J mice were inoculated with 0.5 /¿molof phospholipid (0.01 nC\ of [14C]DSPC) in a volume of 200 ^l of PBS via

a lateral tail vein. Four hr after injection, the mice were killed by cervicaldislocation, and the lungs, liver, and spleen were dissected out and

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f .-"

weighed. A sample of blood was placed in preweighed heparinized glasstubes. Weighed portions of the tissues were digested in 1 ml of Protosol(New England Nuclear) for 24 hr at 50°and placed in 20 ml of Aguasol

(New England Nuclear), and the radioactivity was determined. All countswere corrected for quenching.

In Situ Activation of Mouse Alveolar Macrophages by Freeze-DriedLiposomes Containing MDP-GDP. C57BL/6J mice were inoculated viaa lateral tail vein with freeze-dried liposomes (0.5 ^mol of DSPC:PS; 7:3molar ratio), freeze-dried liposomes admixed with 10 /¿gof MDP, orfreeze-dried liposomes containing 10 /¿gof MDP-GDP in a 200-Ml volume

PBS. Alveolar macrophages were harvested 24 hr later, using theprocedure described for the harvesting of F344 alveolar macrophages,by washing the lungs with 9 x 1 ml of warm PBS. After washing in MEM-FCS, 5x10* macrophages were placed in the wells of microtiter plates,and 5 x 103 [125l]ldUrd labeled B16-BL6 target cells were added. Cocul-

ture was carried out for 96 hr, and the cytotoxicity activity was determined as described above.

Treatment of Experimental B16-BL6 Métastasesin Situ with Liposomes Containing MDP-GDP. B16-BL6 tumor cells in exponentialgrowth phase were harvested by trypsinization, washed by centrifuga-tion, and resuspended in PBS to give 2.5 x 105 viable tumor cells/ml.Five x 104 viable tumor cells in a 200-/J volume were injected via a

lateral tail vein into C57BL/6J mice. Each mouse was treated by i.v.injection with 0.5 ¿¡molof DSPC:PS (7:3 M ratio) freeze-dried liposomescontaining 10 ¿igof MDP-GDP in a volume of 200 n\ PBS on Days 3, 5,

7, 9, and 12 following tumor cell injection. On Day 21, the mice werekilled by cervical dislocation, and the number of pulmonary métastaseswas recorded using a dissecting microscope. Control liposomes wereprepared by omitting MDP-GDP. The effect of treatment on the numberof métastaseswas analyzed using the Mann-Whitney U test.

RESULTS

Stimulation of Macrophage Cytotoxic Activity by Conventional Liposomes Containing MDP or MDP-GDP. Preliminary

studies were carried out to determine the initial incorporationand subsequent retention of MDP in DSPC:PS liposomes. Incorporation studies showed that 0.8 to 1.5% MDP (as [3H]MDP)

were incorporated within 6 to 8 nmol of recovered phospholipid.Subsequent retention studies showed that incorporated MDPwas rapidly lost from DSPC:PS liposomes when incubated at37°(Chart 1). The half-life for retention in MEM-FCS was 5 hr,

with greater than 90% loss occurring with 24 hr. The incubationof liposomes containing encapsulated MDP in serum-free MEM

gave similar retention characteristics (Chart 1). It was observedthat, during the centrifugation steps necessary to remove unen-capsulated MDP, approximately 10 to 15% of the original phospholipid, as measured by [14C]DSPC, was lost. The ability of

such liposomes to stimulate macrophate cytotoxic activity invitro was compared with liposomes containing MDP-GDP. Table

1 shows that liposomes containing MDP significantly stimulatedmacrophage cytotoxic activity at nominal concentrations of MDPat 0.1 and 1 ^g/ml. Control liposomes had no activity under theexperimental conditions. The incorporation of MDP-GDP in lipo

somes resulted in enhanced cytotoxic activity when comparedwith liposomes containing MDP. This enhancement was significant at the 2 highest doses used (p < 0.001). Liposomescontaining MDP or MDP-GDP were both more effective than freeMDP in stimulating macrophage cytotoxic activity (700- and7000-fold, respectively, on the basis of MDP equivalents).

Stimulation of Macrophage Cytotoxic Activity by Freeze-Dried Liposomes Containing MDP-GDP. Freeze-dried liposomes containing MDP-GDP were highly effective in stimulating

100 —

5 10 15 20 25

Incubation time (hr)

Chart 1. Retention of MDP in DSPC:PS (7:3 molar ratio) liposomes. Liposomescontaining MDP spiked with N-acetylmuramyl-L-[6-3Hj]alanyl-D-isoglutamine wereincubated in MEM or MEM-FCS at 37°.At various times, aliquota were removed,and the retention of MDP determined as described in "Materials and Methods."

S.D. from the mean retention was always <5%. O, liposomes incubated in MEM;•,liposomes incubated in MEM-FCS.

macrophage cytotoxic activity (Table 2). Examination of thedegree of cytotoxicity after 72- or 96-hr coculture of pretreatedmacrophages and B16-BL6 target cells showed that maximum

cytotoxic activity was obtained after prolonged coculture. Nocytotoxic activity was observed at 24 or 48 hr of coculture (datanot shown). The ability of free MDP to stimulate macrophagecytotoxic activity followed that of liposomal MDP-GDP in that

enhanced activity was observed after prolonged coculture. Theaugmentation in cytotoxic activity between 72 and 96 hr ofcoculture was, however, less than that observed with liposomalMDP-GDP (1.5-fold augmentation for MDP; 2.5-fold augmentation for liposomal MDP-GDP). The freeze-dried liposomes containing MDP-GDP were considerably more effective in stimulatingmacrophage cytotoxic activity than free MDP (50,000-fold on the

basis of MDP equivalents).The incorporation of MDP-GDP into liposomes was verified by

means of a monoclonal anti-MDP antibody. Conventionally prepared or freeze-dried liposomes were agglutinated by anti-MDPantibody, but not by anti-DNP antibody, for at least 3 monthsafter preparation. Incubation of the liposomes in MEM-FCS at37°for 72 hr had no effect on agglutination, indicating little orno loss of MDP-GDP.

Effect of Liposome Concentration on the Stimulation ofMacrophage Cytotoxic Activity by Freeze-Dried LiposomesContaining MDP-GDP. The effect of liposome concentration on

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Table 1Effect of liposome-incorporatedmuramyl dipeptide on macrophagecytotoxic

activity

Macrophagetreatment"None.

B16-BL6 celtsaloneMEM-FCS

ControlliposomesLjposomal

MDP8Liposomal

MDP-GDPMDP'Concentration

(¿.g/ml)•1.00.10.0081.00.10.0110033113.8Residual

radioactivity0

(cpm)2218±87e2257

±1552179 ±79(3.5)"1290

±87(43)1483±275(34)1897±323(16)336

±99(85)893±164(61)1674±115(26)1501

±103(34)1629±313(28)1725±225(24)1876±144 (17)P<0.001<0.05<0.001<0.001<0.01<0.01<0.05<0.05<0.05

* Rat alveolar macrophages (5 x 104) in a 20XM volume were pretreated with

DSPC:PS liposomes (80 nmol of phospholipid), DSPC:PS liposomes containingMDP or MDP-GDP, or free MDP for 24 hr.

0 Cytotoxic activity was determined after 96 hr coculture of macrophages and 5x 103 |'25l|ldUrd-labeled xenogeneic B16-BL6 target cells.

c Mean ±S.D." Numbers in parentheses, percentage of cytotoxioity.8 Concentration of liposomal MDP or MDP-GDP.' Concentration of free MDP.

the stimulation of macrophage cytotoxic activity by freeze-driedliposomes containing MDP-GDP is shown in Table 3. At a lipo

somal concentration of 400 nmol of phospholipid per ml (80nmol/5 x 104 macrophages), liposomal MDP-GDP was maximally

active at a concentration of 0.009 ng/m\ (equivalent to 0.0045/¿gMDP/ml). Reducing the liposome concentration to 40 nmol ofphospholipid per ml (8 nmol/5 x 104 macrophages) resulted in a

diminution of the cytotoxic activity at comparable levels of MDP-

GDP. This is illustrated by the activity at 400 nmol of phospholipidper 0.09 fig of MDP-GDP, 90% cytotoxicity; and the activity at40 nmol phospholipid per 0.09 //g of MDP-GDP, 41% cytotoxic

ity. Increasing the phospholipid concentration above 400 nmol/ml resulted in a detachment of the macrophages with a resultantloss of cytotoxic activity. Examination of the activity of liposomes

containing MDP-GDP with the activity of free MDP (Tables 1 and2) again showed that the freeze-dried liposomes containing MDP-

GDP were much more effective in stimulating macrophage cytotoxic activity (100,000-fold more active on the basis of MDP

equivalents).Organ Distribution of Freeze-Dried Liposomes Containing

MDP-GDP. The organ distributionof freeze-dried liposomes containing MDP-GDP in mice was determined in order to identify the

DSPC:PS ratio giving efficient pulmonary localization. The results(Table 4) show that DSPC:PS liposomes prepared at a molarratio of 7:3 are efficiently localized within the lungs. Reducingthe DSPC:PS ratio to 7:0.3 or 7:0 resulted in a significantly lowerpulmonary localization of the liposomal preparations. A significantreduction in the localization of the liposomes in the spleen wasalso observed when the molar proportion of PS was reduced(Table 4).

In Situ Activation of Mouse Alveolar Macrophage CytotoxicActivity by Freeze-Dried Liposomes Containing MDP-GDP.The previous experiment had shown that freeze-dried liposomes

prepared from DSPC:PS at a 7:3 molar ratio and containingMDP-GDP were efficiently localized within the lungs. The ability

of such liposomes to activate alveolar macrophages to a cytotoxic state was therefore determined. The injection of freeze-dried liposomes containing MDP-GDP, followed by harvesting of

alveolar macrophages, resulted in the induction of cytotoxicactivity towards B16-BL6 target cells in vitro (Table 5). Control

liposomes or liposomes admixed with MDP had no effect on thesubsequent cytotoxic activity of such macrophages. These results, in conjunction with the organ retention studies, wouldindicate that pulmonary localization of small doses of MDP-GDPin liposomes (<1 n<3of MDP-GDP) are sufficient for the induction

of alveolar macrophage cytotoxic activity.Eradication of Experimental Métastasesby Freeze-Dried

Liposomes Containing MDP-GDP. The previous experimentsshowed that freeze-dried liposomes containing MDP-GDP are

efficiently localized in the lungs, and that such liposomes canactivate alveolar macrophages. The ability of such liposomes toreduce pulmonary metastatic burden of C57BL/6J mice inocu-

Tabte2Effect of freeze-dried liposomescontaining MDP-GDPon macrophagecytotoxic activity

Macrophagetreatment"None,

B16-BL6 cells aloneMEM-FCS

ControlliposomesUposomal

MDP-GDP8MDF/Concentration

(Mg/ml)18.0

0.90.09

0.009500

10010

1.00.1Residual

radioactivity"(cpm)72

hr2562±82°

2752 ±1262626 ±94(Bf1838

±168 (32)1756 ±48 (36)1222 ±52 (56)2094 ±338(24)1384

±113 (52)1870 ±288 (32)2540 + 148(11)2650 ±208 (4)2662 ±100 (3)P<0.001

<0.001<0.001<0.05<0.001

<0.01<0.0596

hr2331

±1031945 ±2171877 ±160(3.5)370

±112 (81)312 ±67 (84)310 ±83 (84)856 ±149(56)606

±153 (69)1034 ±141 (47)1055 ±37 (45)1259 ±284 (35)1428 ±88 (26)P<0.001

<0.001<0.001<0.01<0.001

<0.01<0.01<0.05<0.05

" Rat alveolar macrophages (5 x 104) in a 200-/J volume were pretreated with DSPC:PS liposomes (80 nmol

of phospholipid), DSPC:PS liposomes containing MDP-GDP, or free MDP for 24 hr.Cytotoxic activity was determined after 72- or 96-hr coculture of macrophages and 5 x 103 [125l]ldUrd-

labeled xenogeneic B16-BL6 target cells.0 Mean ±S.D." Numbers in parentheses, percentage of cytotoxicity.8 Concentration of liposomal MDP-GDP.' Concentration of free MDP.

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Table 3Effect of liposome concentration on the induction of macrophagecytotoxic activity by freeze-dried liposomes

containing MDP-GDP

Macrophagetreatment"None,

B16-BL6 cells aloneMEM-FCSControl liposomesConcen

tration(xg/ml)cpm

of residualradioactivitySOnmol

phospholipid2076±202"

2214 ±1982042 ±56 (8fSnmol

p phospholipid1962

±1341986 ±361900 ±98(4)P

Uposomal MDP-GDP 18.0 420 ±168(81) <0.0010.9 494 ±202 (78) <0.0010.09 228 ±202 (90) <0.0010.009 416 ±32(81) <0.001

1.80.090.0090.0009

824 ±162 (63)1170 ±182(41)1662 ±112(16)1940 ±46(1)

<0.001<0.01<0.01

* Rat alveolarmacrophages(5 x 104)in a 200-/J volume were pretreated with DSPC:PSliposomes(80 or 8nmol of phospholipid),or DSPC:PSliposomescontaining MDP-GDPfor 24 hr.

Mean ±S.D.°Numbers in parentheses, % of cytotoxicity. Cytotoxic activity was determined after 96-hr coculture of

macrophagesand 5 x 10' [125l]ldUrd-labeledxenogeneicB16-BL6 target cells.

Table 4Organ distribution of freeze-dried liposomes containing MDP-GDP

Groupsof 5 C57BL/6 micewere given injectionsof [14C]DSPC-labeledDSPC:PSliposomes(0.5 /imo! of phospholipid)containing MDP-GDP(10 «g)in a volume of200 ni via a lateral tail vein. The mice were sacrificed 4 hr later, and the retentionof the "C-label by the various tissues determined as described in "Materials andMethods."

DSPC:PS(molarrat«)*7:3

7:0.37:0%

of Retention ofphospholipid1'Lung8.6±1.4c-d

1.2 ±0.11.4 ±0.2Liver45.0

±8.437.3 ±5.042.3 ±1.6Spleen2.4

±0.1"2.0 ±0.2a

1.6 ±0.1Blood2.9

±1.33.9±0.8

4.3 ±1.6" DSPC:PSMratio was used to prepare the liposomes." The retention of "C label is expressed as the percentage of radiolabel per

organ. The results for whole blood are expressed as the percentageof radiolabelper ml of blood.

c Mean ±S.D." Significantlydifferent from DSPC liposomes (7:0 Mratio);p < 0.05.

TablesIn situ activation of murine alveolar macrophagesby freeze-dried liposomes

containing MDP-GDPIn situ treatment* cpm of residual radioactivity

None, B16-BL6 cells alonePBSControl liposomesControl liposomes + MDPLiposomal MDP-GDP

2032 ±160"

2019 ±1191876±200(7)c

1973 ±94(2)1134 ±103" (44)

a Groups of C57BL/6 mice were treated with DSPC:PSliposomes (0.5 limo»of

phospholipid; 7:3 M ratio), DSPC:PS liposomes admixed with 10 ug of MDP orDSPC:PSliposomes containing 10 ^g of MDP-GDPin a 200-nl volume via a lateraltail vein.Alveolarmacrophageswere harvested24 hr lateras describedin 'Materialsand Methods." After plating 5 x 104macrophages,5 x 103[1Ml]ldUrd-labeledB16-BL6 cells were added, and macrophage-mediatedcytotoxicity determinedafter 96-hr coculture.

6 Mean ±S.D.' Numbers in parentheses, percentage of cytotoxicity."p< 0.001.

lated previously i.v. with B16-BL6 tumor cells was therefore

determined. The results (Table 6) show that repeated doses ofliposomes containing MDP-GDP (0.5 nmo\ of phospholipid per10 ug of MDP-GDP) are able to reduce the metastatic burden ofmice bearing the B16-BL6 tumor. Control liposomes or free MDP(10 ug) had no significant effect on the pulmonary metastaticburden. Examination of the size of the métastases remaining

TableeEffect of freeze-dried liposomes containing MDP-GDPon B16-BL6 métastasesGroups of C57BL/6 mice were inoculatedon Day 0 with 5 x 104B16-BL6 cells

via a lateral tail vein.

Treatment*Control

(PBS)Control liposomesüposomalMDP-GDP,10 MgMDP, 10/igNo.

ofmice15

888Mean

no. ofpulmonary

métastases"31

(19-42)c33(18-50)

8" (2-14)35(22-53)

* Control DSPC:PSliposomes (0.5 ^mol of phospholipid), DSPC:PSliposomescontaining 10 ^g of MDP-GDPor 10 /¿gof free MDP in a 200-jil volume of PBSwere injected via a lateral tail vein on Days 3,5, 7,9, and 12 following the injectionof B16-BL6 cells.

6 The number of pulmonary métastaseswas determined on Day 21 followinginjectionof B16-BL6 cells.

' Numbers in parentheses,range." Significantly different from control-treated mice (Mann-Whitney U test); p <

0.001.

after treatment showed that small (<1 mm diameter) and intermediate (<2 mm) métastases had been eradicated, but thatthere was no effect on large métastases(>2.5 mm).

DISCUSSION

The use of lipophilic derivatives for the enhanced liposomalincorporation and retention of compounds having a high degreeof hydrosolubility was first described in detail for the liposomalencapsulation of cortisol esters (19,26). The possible advantageof using phospholipid or glycolipid esters for liposomal incorporation was initially proposed in studies on the action of liposomal-

encapsulated antiarthritic drugs (17). Such approaches wouldappear to have considerable application for the augmentation ofmacrophage cytotoxic activity by muramyl peptides.

MDP has been shown to directly augment macrophage cytotoxic activity in vitro (32), but the observation that MDP is rapidlyexcreted in vivo (18) poses the problem of a sufficient exposureof the macrophage effector cells for the induction of cytotoxicactivity. The ability of cells of the reticuloendothelial system tointeract with liposomes provides a means of selectively targetingthe muramyl peptides to cells of the monocyte-macrophage

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ACTIVATION OF ALVEOLAR MACROPHAGE TUMORICIDAL ACTIVITY

lineage.A number of immunomodulatory agents, i.e., macrophage

activating factor, lipopolysaccharide, and MDP, have been encapsulated within liposomes and shown to be effective in inducing macrophage cytotoxic activity against syngeneic, allogeneic,or xenogeneic tumor cells in vitro (8,12, 25, 27, 28, 30, 31, 32).The optimal liposome composition and structure for efficientinteraction with the macrophage have been determined, and theirlocalization in target organs such as the lung has been extensively studied (6, 21, 23, 25). While the dose effectiveness ofliposome-encapsulated MDP in inducing macrophage or mono-

cyte cytotoxic activity in vitro has been reported (28, 31, 32),until this study, no detailed analysis of the relative efficacy ofliposome-incorporated MDP lipophilic derivatives has been made.

One problem which is often neglected in the description ofconventionally prepared liposomes containing MDP (that is, hy-

dration of dried lipid films) is the time required for their preparation, the reproducibility of composition, and the question of long-

term storage. For these reasons, we have used liposomesformulated by freeze-drying the constituent components (2) followed by a one-step hydration. We have compared their efficacy

with conventionally prepared liposomes using the xenogeneicmacrophage-tumor cell system of rat alveolar macrophage andmurine B16-BL6 tumor cells described by Sonéand Fidler (28).

In contrast to mouse macrophages, rat macrophages wouldappear to require only one signal for the induction of cytotoxicactivity (27), thus providing the opportunity of directly evaluationthe activity of such liposome preparations. The results presentedin this report provide a detailed analysis of the dose effectivenessof such liposomes in stimulating macrophage cytotoxic activity.

The inclusion of MDP into conventionally prepared liposomesresulted in a 700-fold increase in efficacy compared with freeMDP. Analysis of the dose effectiveness of MDP-GDP incorpo

rated into liposomes showed that the lipophilic MDP derivativewas 7000-fold more effective than free MDP. In studies usinganother lipophilic MDP derivative, MTP-PE, it was observed thathydrosoluble MDP or MTP-PE incorporated into liposomes were

equally effective in stimulating macrophage cytotoxic activity(25). Our results would indicate that the inclusion of the lipophilicMDP derivative MDP-GDP into conventional liposomes results in

a significant enhancement of macrophage cytotoxic activity compared with liposomal MDP. That lipophilic MDP derivatives areintrinsically more active than hydrosoluble MDP has been demonstrated in studies on the release of Superoxide anión frommacrophages (12, 15). The use of freeze-dried liposomes re

sulted in a significant enhancement of macrophage cytotoxicactivity compared with liposomal MDP-GDP prepared by conventional means. The augmentation in efficacy (>50 to 100,000-

fold) compared to free MDP indicates the effective concentrationof liposomal MDP-GDP for induction of macrophage cytotoxic

activity is of the order of pg/ml. Such liposome formulationscould be kept in powder form for several months without loss ofactivity.

The poor retention of hydrosoluble MDP in DSPC:PS liposomes illustrates the advisibility of using lipophilic MDP derivatives for the incorporation of MDP in liposomes. The reactivity ofliposomes containing MDP-GDP with monoclonal anti-MDP an

tibody indicated little or no loss of the muramyl dipeptide onprolonged storage of the liposomes after reconstitution.

In contrast to previous reports on the kinetics of macrophage

activation by liposome-incorporated MDP or MTP-PE (25, 28,

32), maximum cytotoxic activity was not observed with liposomalMDP-GDP until 96 hr after the initial treatment. The concept of

a depot of liposomal MDP within the macrophage has beenproposed as being advantageous for cytotoxic activity (25);MDP-GDP would appear to offer some advantage over other

muramyl dipeptide liposome preparations in this respect. Theobservation that alveolar macrophages are capable of exhibitingmaximal cytotoxic activity for at least 6 days after the initialphagocytosis of liposomal MDP-GDP4 supports this supposition.

We believe that a study of the ability of lysosomal enzymes todisrupt and degrade such liposome preparations would providemuch needed information in this area.

The importance of liposome to macrophage ratios for theinduction of cytotoxic activity in vitro by liposome incorporatedmuramyl dipeptide (28) has been confirmed in this study. Ourobservation that high liposome concentrations lead to a detachment of macrophages in vitro4 may have some relevance to

macrophage activation in vivo.One problem in the treatment of malignant tumors is the

production of métastasesdiffering in their response to treatment.Macrophage tumoricidal activity would appear to be indépendantof tumor drug sensitivity, antigenicity, or metastatic potential (31,32). Evidence for the involvement of pulmonary macrophages inthe eradication of murine melanotic métastasesafter the systemic administration of liposomes containing MDP or MTP-PE

has been obtained in several studies (7, 11, 25). In our studies,the use of an experimental B16 melanoma (i.v. injection of tumorcells) was chosen in preference to a spontaneous metastaticmodel (s.c. injection of tumor cells followed by excision of theprimary tumor) because of the reported phenomenon that macroscopic (>1 mm in diameter) métastases do not respond toliposomal MDP treatment (11). In our preliminary experiments,we established that freeze-dried liposomes containing MDP-GDP

were efficiently arrested in the lungs, and that alveolar macrophages from normal mice inoculated i.v. with such liposomeswere cytotoxic towards B16 melanoma cells. The demonstrationthat liposomes containing MDP-GDP were effective in reducing

the pulmonary metastatic burden of mice inoculated with B16melanoma cells validated the supposition that such a liposomeformulation has antitumor activity.

In conclusion, we have demonstrated that a member of a newclass of lipophilic MDP derivatives, MDP-GDP, is a potent acti

vator of macrophage cytotoxic activity in vitro when incorporatedinto liposomes. The use of a freeze-dried procedure for liposome

formulation, which allows identical batches to be prepared andstored in powder form, resulted in a further increase in efficacywhen compared with hydrosoluble MDP. The in vivo antitumoractivity of such liposomes was established using the syngeneicB16 pulmonary metastatic model. Further studies to establishthe dose effectiveness of such liposome preparations in activating alveolar macrophages and in eradicating pulmonary métastases are currently in progress.

ACKNOWLEDGMENTS

We thank George M. Bahr for the samples of monoclonal anti-MDP andmonoclonalanti-dinitrophenylantibodies.

4N. C. Phillips, M. L. Moras, L. Chedid, P. Lefrancier, and J. M. Bernard,

unpublishedobservations.

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1985;45:128-134. Cancer Res   Nigel C. Phillips, Maria L. Moras, Louis Chedid, et al.   DerivativeLiposomes Containing a New Lipophilic Muramyl DipeptideEradication of Experimental Metastases by Freeze-Dried Activation of Alveolar Macrophage Tumoricidal Activity and

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