[CANCER RESEARCH 41, 1602-1607, May 1981]0008-5472/81 /0041-OOOOS02.00

Selective Delivery of Liposome-associated c/s-Dichlorodiammineplatinum(ll)by Heat and Its Influence on Tumor Drug Uptake and Growth1

M. B. Yatvin,2 H. Mühlensiepen,W. Forschen, J. N. Weinstein, and L. E. Feinendegen

Institut fürMedizin. Nuclear Research Center. KFA. D-5170 Jülich,West Germany [H. M., W. P.. L. E. F.¡:Human Oncology Department, University of Wisconsin,Madison. Wisconsin 53792 [M. B Y.]: and Department of Theoretical Biology. National Cancer Institute. NIH. Bethesda. Mary/and 20205 [J. N. WJ

ABSTRACT

In an attempt to optimize the chemotherapeutic treatment ofmouse tumor Sarcoma 180, liposomes containing c/s-dichlo-

rodiammineplatinum(ll) (FDD), having transition temperaturesa few degrees higher than the rectal temperature of mice, wereused in combination with local hyperthermia. The uptake ofradioactive PDD by tumors heated for 1 hr at 42°was almost

four-fold greater when the drug was associated in liposomesthan if administered as free drug. Uptake of liposome-admin-

istered radioactive platinum by liver was twice that obtainedwith free PDD, whereas its incorporation by the kidney was thesame by either method of drug administration.

The effect of various combinations of hyperthermia, drug-

containing liposomes, and free PDD on tumor growth was alsostudied. Treatment with liposome-associated PDD plus localheating resulted in a dose-modifying factor of 7 when comparedwith free drug and no hyperthermia. The dose-modifying factor

was 2.5 when PDD liposomes and heat were compared withinfree drug and heat. Thus, PDD could be specifically releasedfrom liposomes by heat and resulted in both a greater druguptake and a delayed tumor growth following treatment. Potential normal tissue toxicity problems, however, still need to beresolved before clinical application of this combined modalitywill be possible.

INTRODUCTION

To optimize the effectiveness of a given chemotherapeutictreatment of malignant disease, target specificity of the drug isan important goal. Unfortunately, most chemotherapeuticagents interact with nonmalignant tissue, markedly reducingtheir therapeutic effectiveness. For example, although the an-titumor agent PDD3 has a significant activity against a number

of different tumors in animals and cancers in humans, neph-rotoxicity reportedly is the major dose-limiting side effect (3,

10, 19,23,24).In experiments with mice, the dose of PDD required to kill

50% of the animals ranged between 13 and 14 /ig/g bodyweight (23). Using [125l]iododeoxyuridine-labeling techniques,local hyperthermia (42°) for 1 hr had only a small effect onSarcoma 180 growth and rate of loss of 125Iin NMRI mice (30).

When PDD was combined with hyperthermia, its tumor cyto-

toxicity was enhanced under both in vivo and in vitro growthconditions (9, 17).

Liposomes, which are microscopic vesicles composed of

' These studies were supported in part by NIH Grant GM-91846.2 To whom requests for reprints should be addressed.3 The abbreviations used are: PDD, c/s-dichlorodiammineplatinum(ll); DPPC,

dipalmitoylphosphatidylcholine: T,, liquid-crystalline transition temperature;DSPC. Disteroylphosphatidylcholine; DMF, dose-modifying factor.

Received March 18, 1980; accepted January 22. 1981.

phospholipid bilayers enclosing aqueous compartments, haverecently been the object of much interest for tumor therapy (4,7, 20, 28). A major limitation on their potential usefulness,however, has been target specificity. In an attempt to overcomethat deficiency, we have suggested a combined approach invivo in which drug-containing liposomes having specific lipid

transition temperatures are heated in vivo to obtain a preferential release of drug in a target area (34, 35). In subsequentexperiments using liposomes containing [3H]methotrexate in

combination with local heating of tumor in mice, 4 times asmuch drug was delivered to heated murine tumors as comparedwith unheated tumors (31).

In the current study, we report on experiments in tumor-bearing mice using hyperthermia in combination with liposome-associated PDD. The clinical potential of PDD, which is highlycytotoxic at low concentrations, has been considered in detailin a number of reviews (3, 19, 23, 24). The consensus is thatc/s-platinum compounds are valuable for treatment of certain

human tumors. Its use in tumor therapy could be further facilitated if normal tissue toxicity could be either reduced or eliminated by preferentially releasing the drug in a target area usingliposomes. Its high activity could be clinically relevant becauseof the limited carrying capacity of small liposomes. This reportdescribes the current status of our efforts to obtain bettertherapeutic ratios in treating murine tumors with PDD liposomesand local hyperthermia.

MATERIALS AND METHODS

Drugs and Chemicals. The PDD compound and the lipidsDPPC (T,, 41 °)and DSPC (T,, 54°)were purchased from Serva,

Heidelberg, West Germany. Radioactive platinum was produced by activation of the PDD by thermal neutron flux of 6.5x 1010 neutrons sq cm/sec over a period of 12 hr in the Dido

research reactor at the KFA Jülich.The temperature in theirradiation position was maintained below 40°. Irradiated PDD

was mixed with unirradiated PDD (1:2) for a final concentrationof 2 mg/ml. Experiments were always carried out 5 days afterneutron irradiation to allow the radioactivity to decay primarilyto 195mpt(half-life, 4.1 days). Platinum purity was determined

by thin-layer chromatography (33) [acetone:water (7:3)]. Acti

vation did not influence PDD purity in any apparent way, andsimilar single spots were obtained both before and after neutronirradiation.

Preparation of Liposomes. Small liposomes with a maximaloutflow of drug at 42°were produced by sonication of DPPC:

DSPC (7:1). Prior to sonication, the lipids were dissolved inchloroform, dried onto the walls of a glass vial under a streamof nitrogen, and kept in a high vacuum overnight. Six ml of thePDD solution were added to 100 mg of lipid and vortexed to asuspension at about 50°.The suspension was then sonicated

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Delivery of Liposome-associated PDD

for 1 hr at 50°with the microtip of a Branson sonifier (Branson

Instruments, Danbury, Conn.) under a stream of nitrogen. Thesonicate was rapidly cooled in an ice bath, and any titaniumfragments released from the microtip during sonication wereremoved by centrifugation (2 x 103 x g for 10 min). Thesupernatant was then stored at 6°overnight. It became pro

gressively more turbid over the storage period as a result of aslow increase in vesicle size (25). Immediately before use, thefree PDD was removed by passing the liposomes through aSephadex G-50 (fine) column (1.8 x 30 cm). The large liposomes were removed by ultracentrifugation at 105 x g for 30

min (11, 22). The resulting optically clear supernatant wasconsidered to contain only small liposomes. Large liposomeswere obtained by resuspension of the precipitate in 167 rriMNaCI:6 mw KCI adjusted to pH 7.4 and vortex mixing atmaximum speed. The amount of the PDD in the sonicatingsolution associated within small liposomes was 3.9 ±0.5% oftotal PDD, and the injection doses were calculated from radioactivity measurements with scintillation counting. The loss oflipids by handling, separation, etc. (about 40%) and the injected dose were analyzed by measurement of light scatter at410 nm (32) in a spectrophotometer (Carl Zeiss, Oberkochem,West Germany).

Determination of the Maximal PDD Release Temperature.Shown in Chart 1 is the temperature dependence of PDDrelease from small liposomes either in the presence of 10%mouse serum or with 5 x 105/ml Sarcoma 180 cells. One-mi

suspensions of vesicles (6 mg lipids) were placed in a waterbath and heated for a total of 3 min. The temperature in theliposome sample was monitored continuously with a thermistorprobe and, starting from 0°, reached the test temperaturesbetween 37 and 44°within approximately 90 sec. The sample

was then kept at the respective test temperatures for an additional 90 sec. The maximum release is at 42°,the apparent Tt

of these liposomes. At the T,, the lipids in the bilayer are in arapid state of flux, and permeability is maximal. When the Tt isexeeded, leakage from the aqueous compartment is decreasedbelow this maximum. The phenomenon of increased release ofsolution close to the transition temperature in vitro has beendescribed previously (8, 21). Using carboxyfluorescein-con-

100

LU(/)<LU

OoQ_

50

O 37 39 41 43

TEMPERATURE

Chart 1. Temperature dependence of radioactive PDD release from smallliposomes composed of DPPC:DSPC (7:1). A 1-ml suspension of liposomescontaining 1.8 /ng PDD and 6 mg lipids was mixed with either 10% mouse serum(O, •)or 5 x 105 Sarcoma 180 tumor cells (x), placed in a water bath, andheated from 0°to between 37 and 44°. The temperature in the sample was

monitored continuously during heating which reached its final temperature in 90sec. The induced release of PDD was determined by passing the sample througha Sephadex G-50 (fine) column, counting the PDD activity in the recoveredliposomes, and subtracting it from the initial counts. Open and closed circles areduplicate experiments.

taining liposomes, we obtained similar results in earlier studies(33, 34). Release of PDD was determined by free-drug retention on chromatography columns (10-ml syringes) of Sephar-dex G-50 (fine). Essentially identical results were obtained in

the presence of mouse serum or tumor cells with 40 to 60% ofthe PDD being released from liposomes at 42°during the 90-

sec heating interval.Animals and Tumor. Sarcoma 180 tumors were grown s.c.

in the hind leg of 10- to 12-week-old female NMRI mice byinjecting 3 x 105 cells. The tumor was allowed to develop for

7 days before use. Tumor volumes were calculated from calipermeasurements with the sizes ranging between 0.70 and 1.0 cucm.

Treatment and Experimental Design. Hyperthermia over arange of 40 to 44°was applied to the tumors by immersion of

the tumor-bearing legs of unanesthetized mice in a water bath

for a period of 60 min after injection of PDD in its various forms.For heating, the tumor-bearing leg is fixed in a special hyper-

thermia device with Tesa tape (29). The tumor volume experiments were run at 42°. In those experiments, the temperature

in the center of the tumor was measured with a small, 0.8-mm

diameter, Thermistor probe (Tastomed P, Braun Electronic,Frankfurt, West Germany). The tumor temperatures rose within5 min from approximately 34 to 41.5° and came up to 41.9°within 10 min. Rectal temperature increased from 37°within 5min to 39 to 39.5° and varied by less than 0.5° during the

heating (29). After preheating the tumor for 5 to 10 min, freedrug or drug-containing liposomes were injected into the tail

vein. For clearance measurements, blood was collected atdifferent times after injection from the tail vein with capillarytubes. The activity in the whole body, blood, various organs,and tumors was measured in 4- x 4-well-type Nal scintillation

counters.

RESULTS

Drug Clearance Studies. Shown in Chart 2 is the rate ofPDD clearance from the blood when injected either in free formor associated in liposomes. The associated PDD was clearedin 2 phases, an initial rapid loss and a slower period of decline.The result can be fitted to functions of the form

A = e-e-0693-'"'' + c.e-°693-'/r*

AO= B + C

where A is the relative blood level, A0 is 100%, and B, C, 7",,

and T2 are constants calculated graphically. Most of the freePDD was cleared from the blood within 5 min in animals withheated tumors. Local tumor hyperthermia had only a slightinfluence on the clearance rate of liposomal PDD. From earlierstudies with methotrexate, a more rapid clearance from theblood was expected when the liposomes passed through theheated tumor area (31). The difference is probably the resultof PDD having a greater affinity for lipid. Based on the calculated volume of the internal aqueous compartment of smallliposomes, we estimated that, under our experimental conditions, less than 0.25% of the drug would have been associated;however, we found approximately 20 times as much associatedwith the liposomes. Depending on how the drug was partitionedinto the lipid bilayer, it might or might not be expected to exitfrom the liposome bilayer slower than from the aqueous com-

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M. B. Yatvin et al.

10

20 40 60 80MIN AFTER INJECTION

120

Chart 2. Clearance of PDD from the blood after i.v. injection either as freedrug or associated in DPPC:DSPC (7:1) liposomes. In each liposome experiment,1.2 /ig PPD per g body weight and 0.13 mg lipid per g mouse body weight wereadministered. The same quantity of free drug was given. The treatment groupswere as follows: x, small liposome with tumor unheated (n = 9); •,smallliposome with tumor heated (n = 9); O, large liposome with tumor unheated (n= 6); D, free drug with tumor unheated (n = 3). In the free-drug experiments,1.2 fig PDD per g body weir,ht was also injected. Bars, S.D.

partment (27). In the case of PDD, it apparently exited moreslowly which could account for the reduced rate of clearancefrom the blood. As expected from the studies of Juliano andStamp (11), we found that the rate of clearance of largeliposomes from the circulating blood was more rapid than thesmall liposomes (Chart 2). In the absence of heating, tumoruptake of PDD entrapped in these large liposomes was onlyone-fourth that obtained when small liposomes were injected,

whereas, in both liver spleen, the uptake of radioactivity wasenhanced 3-fold (Table 1). In contrast, the uptake of radioac

tivity by the kidney after injection of large liposomes was onlyone-third that obtained after administration of PDD either asfree drug or as small liposomes with or without heating thetumor.

Effect of Temperature on Tumor Uptake of Drug. Theeffect of elevated temperature on PDD uptake by tumors wasstudied for a 24-hr period after heating. To exclude the possi

bility that differences in the uptake of radioactive platinumresulted from systemic rather than local effects of heating,experiments were performed with mice carrying tumors in bothhind legs (double-tumor mice). In these experiments, one tumor

was heated while the other served as its control. Tumor heatinghad no measurable effect on the uptake of free PDD. Theradioactivity remained nearly constant over the 24-hr postinjection period. After injection of liposome-associated PDD,drug accumulation in 42°-heated tumors reached a maximum

of 8.2% within 4 hr after injection (Table 1). In double-tumor-

bearing mice, uptake of associated PDD was higher in theheated tumor than in the unheated control tumor. Likewise, insingle-tumor-bearing mice, uptake was higher in the heated

tumor when it was compared with mice given similar injectionand bearing single unheated tumors. The ratio of uptake ofassociated to free PDD in 42°-heated tumors was 2.2 at the

end of heat treatment, rising to 3.8 at 4 hr and falling back to2.6 by 24 hr (Chart 3/4). Similar though less striking results

Table 1

Organ radioactivity 4 hr postinjection

Organ radioactivity is presented as percentage of total injected dose.

TumorLiverSpleenKidneyFree

PDD2.4±0.5a

9.9 ±2.00.5 ±0.23.6 ±0.4Unilamellar

liposomes4.4

±0.717.8 ±2.0

0.8 ±0.24.1 ±0.2Unilamellar

liposomes +

42°8.2

±0.519.0 ±3.0

0.8 ±0.24.4 ±0.4Multilamellar

liposomes2.1

±0.655.4 ±5.0

2.4 ±0.41.3 ±0.3

3 Mean ±S.D. (n = 6).

¿00

300 -

200

100

300

4 24HOURS POST INJECTION

200

100

HEATED LIPOSOMESUNHEATED LIPOSOMES

1 4 24HOURS POST INJECTION

Chart 3. Incorporation of neutron-activated PDD into heated Sarcoma 180tumors 1, 4, and 24 hr after tail vein injection of free or associated drug. Heatedtumors were maintained at 40°O (n = 4); 42°(•)(n = 12); and 44°(O) (n =

4) for 5 to 10 min prior to and 1 hr postinjection. Tumor radioactivity is notcorrected for blood PDD activity. In A, the percentage of increase in tumor uptakeis determined between liposome-injected animals whose tumors were heated andfree PDD-injected animals with heated tumors. In B, the percentage of increasein tumor uptake is determined between liposome-injected animals whose tumorswere heated and liposome-injected animals in which the tumors were unheated.

were obtained when heated-liposome to unheated-liposome

tumor uptake ratios were determined (Chart 36). The greateruptake of associated PDD is most likely due to an increasedlocal availability of PDD as a result of a hyperthermia-induced

release of PDD from liposomes at the target site.In contrast to the data presented in Chart 1 on release of

PDD from liposomes, PDD uptake by tumor was essentially thesame after heating for 1 hr at either 42 or 44°. A number of

possible explanations could account for this apparent discrepancy. First, the temperature in the heated tumor is probablynot uniform throughout under water bath heating conditions.Thus, liposomes could be exposed to a range of temperaturesas they passed through the heated area. In tumors heated toeither 42 or 44°, at least some parts are likely to be close to

the presumptive T,. In addition, blood, when it enters the tumor,is around 37°and is heated as it traverses the tumor. Thus,even in regions where the tissue is 44°,the temperature of the

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Delivery of Liposome-associated FDD

blood would be slightly lower. Another possibility is that, eventhough less PDD may be released from liposomes at 44°

(above the 7,), it is possible that heating the cells at the highertemperature resulted in changes in the tumor cell membranesallowing more efficient uptake of the drug by the tumor.

Influence of Various Combinations of Free PDD, Liposome-

associated PDD, and Heat on Tumor Growth. To study theeffect on tumor growth, free PDD was injected over a doserange of 1.5 to 10.6 /¿g/gbody weight, and liposome-associ-

ated PDD was injected at 0.7 and 1.2 jug/g body weight tomice bearing single tumors. Chart 4/4 shows the increasingtumor volume with time after injection of 1.5, 5.3, and 10.6 ¡igPDD per g body weight with and without tumor hyperthermia.Shown in Chart 46 are the patterns for unheated and heatedcontrol and liposome-injected animals (1.2 fig PDD per g and0.13 mg lipid per g body weight). One hr of 42°hyperthermia

had very little effect on the rate of tumor growth (Chart 46).The greatest inhibition of tumor growth was obtained in theanimals receiving 10.6 jug of PDD per g body weight in combination with hyperthermia. This group, however, also had thegreatest mortality rate, with all the animals dying by Day 5.

The combination of liposome-associated PDD and heat was

more effective per amount of PDD in delaying tumor growththan when tumor heating was combined with administration offree PDD (Chart 4, A and ß).The treatment-induced tumor

growth delay data for the different treatments are shown inChart 5. Growth delay is calculated from Chart 4. An exampleof how this is done is presented in Chart 48. Growth delay isdefined as follows: the difference in days between the time

5 10 15

DAYS AFTER TREATMENT20

Chart 4. Growth of Sarcoma 180 tumor as measured by an increase in relativetumor volume after injection of either free or liposome-associated PDD alone orin combination with hyperthermia (42°) 5 to 10 min before and 1 hr after drug

injection (n = 8). Various levels of free PDD were injected i.v. in 0.5 ml.Liposomes containing 1.2 fig PDD and 0.13 mg lipids per g body weight werealso injected i.v. in 0.5-ml aliquots either as a single therapy or in combinationwith hyperthermia. The free PDD groups in A are as follows: control (x); 1.5 figPDD per g (A); 1.5 fig PDD per g plus heat (A); 5.3 fig PDD per g (O); 5.3 fig PDDper g plus heat (•);10.6 fig PDD per g (D); 10.6 fig PDD per g plus heat {•)Thecontrol and liposome-associated PDD groups in B are: control (A); heat only(•);1.2 fig PDD-liposome per g (x); 1.2 fig PDD-liposome per g plus heat (O).

5 10

PDD/g MOUSE

Chart 5. Treatment-induced tumor growth delay is calculated as indicated inTable 2. Hyperthermia alone (60 min, 42°) induced tumor growth delay of about

0.5 days. There was no significant difference in the tumor growth delay obtainedin animals given injection of free PDD and those receiving injection of PDD-

liposomes and in which tumors were not heated. The latter group is therefore notshown. Open symbols, 5 animals/group; closed symbols, 8 animals/group. Thedata point for animals receiving 0.7 fig/g mouse is obtained from growth delaydata not shown in Chart 48. LIP, lipids; HYP, hyperthermia.

Table 2Dose-modifying factors

Treatment comparisonratiosPDD-liposomes/PDD

PDD-liposomes + 42°/PDD-lipo-

somesPDD + 42°'/PDDPDD-liposomes + 42°/PDDPDD-liposomes + 42°/PDD + 42°TumorRadioactive

uptake"1.8

±0.4c(1.6)d

2.0 ±0.4(2.7)1.0

±0.23.8 ±0.5 (3.5)3.7 ±0.6Growth

delay6ee2.5

±1.57.0 ±3.62.5 ±1.4

DMF values for the tumor uptake are calculated from the measured tumoractivity 4 hr after injection of labeled PDD (n = 4 to 12). The injected dose iscontrolled by activity measurements.

b DMF values for tumor growth delay are calculated from Chart 5 and obtained

as indicated in the following example. The growth delay in days for a givenquantity of liposomes encapsulating PDD (0.7 or 1.2 fig/g mouse) plus hyperthermia is compared to the dose of PDD alone or PDD plus heat needed toproduce the same growth delay. The DMF is obtained by dividing the formervalue by the latter.

c Mean ±SD.d The blood activity in the tumor is calculated from the blood volume (7% of

the weight) and from the blood PDD 4 hr after injection. Numbers in parentheses,data corrected for blood radioactivity.

e The results from the unheated PDD-liposomes were not shown in Chart 5 as

they fall on the shoulder of the PDD-effect curve and were not significantlydifferent from it. Thus, the growth delay of DMF's involving these groups is not

presented in Table 2.' Hyperthermia in these studies consisted of exposing the tumor to 42°for 1

hr after a preheating period of 5 to 10 min to stabilize the temperature in theheated area. If not designated 42°, the tumors were unheated and had coretemperatures of 34°.

required for the treated tumor to double its initial volume relativeto the control doubling time. Analysis of the results using theisobologram method introduced by Steel and Peckham (25)suggested a supraadditive effect for the combination of hyperthermia with either free PDD or liposome-associated PDD intreatment-induced tumor growth delay of 1.5 and 3.0 days.

The latter combination had the greatest effect.DMF's. The calculated DMF for both treatment-induced tu

mor growth delay and uptake of radioactivity for the varioustreatment combinations is shown in Table 2. The uptake ofradioactive PDD by tumors heated for 1 hr at 42°was increased

almost 4-fold when the drug was associated in liposomes

compared to the level obtained when it was administered asfree drug. The uptake DMF was 2.0 when PDD-liposomes andheat were compared to unheated PDD-liposomes (Table 2).The 10.6-jiig heated PDD group is not used in the calculation.In the groups treated with liposome-associated PDD, we cannot

see a cytotoxic effect, but, with a higher PDD dose, the mice

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M. B. Yatvin et al.

lose weight shortly (1 to 2 days) after treatment.Even more striking results were obtained when the DMF

values for treatment-induced tumor growth delay were com

pared for the various treatment groups. The free FDD plushyperthermia groups had a DMF of 2.5 compared to PDD only.This induced growth delay occurred despite the fact that therewas no difference in uptake of radioactive PDD between thesegroups. The greatest growth delay DMF values (7) were obtained when the heated PDD-liposome group was compared toeither the unheated PDD-liposome group or the unheated, free

PDD group. A comparison between free drug treatment andPDD liposomes, when both were heated, resulted in a growthdelay DMF of 2.5 (Table 2).

DISCUSSION

Cancer chemotherapy, ideally, aims at targeting antineoplas-

tic drugs to the tumor with little or no incorporation by cells ofnormal tissue. The strategy of using liposomes to attain thatgoal has been repeatedly discussed (see reviews in Refs. 6,14, 15, and 18); however, successful drug targeting usingliposomes without normal tissue involvement remains elusive.In an attempt to resolve some of the problems in targeting, wehave recently suggested that liposomes could be combinedwith local hyperthermia to achieve preferential release of adrug in a target area (34, 35). The drug-containing liposomes

are formed by a phospholipid mixture that exhibits a gel stateto T, a few degrees above the physiological temperature in therange easily obtainable by local hyperthermia. It was postulatedthat such liposomes would remain reasonably stable in thevascular system at normal body temperature (T < Tt), and,when passing through the heated area, they would releasetheir contents when the T, is attained (34, 35). This approachwas tested in an experimental system using the highly water-

soluble drug methotrexate (32). More than 4 times as muchradioactive methotrexate was accumulated in murine tumorsheated to 42°compared with that bound in unheated controltumors after i.v. injection of drug-laden liposomes.

Likewise, with PDD, a preferential release was obtained fromliposomes around their T, (Chart 1). Our earlier studies (31, 34,35) support best the hypothesis that local heating leads toselective drug release from liposomes, resulting in an increasedlocal concentration of polar drugs (hydrophilic) in the heatedarea. In the case of methotrexate, the drug then apparentlyentered the Lewis lung tumor cells by normal transport mechanism (31). In the same study, it was also found that, whenDPPC:DSPC liposomes were labeled with [14C]DPPC, the ra

dioactive uptake ratio for heated and unheated tumors averaged 1.9. Although considerably less than the methotrexateincorporation ratios, it is suggestive that mechanisms otherthan selective release at the T, may be operative in accountingfor the greater uptake of liposome-associated drug in the

heated tumor. The enhanced uptake of lipid from labeledliposomes noted above could be explained in a number ofdifferent ways (i.e., fusion, endocytosis, or lipid exchange)(32). Furthermore, the current studies indicate that PDD, eitherpartitioned into or associated with the lipid bilayer, apparentlycan also be released from liposomes by heat. As shown inChart 1, 40 to 60% of the PDD associated with the liposomesis released when they were exposed to 42°for 90 sec. Possi

bly, an increase in partitioning of the drug into the lipid bilayer

and its release by prolonged heating may explain this phenomenon. Although liposome retention time within the heated tumoris not likely to approximate 90 sec, drugs may also be releasedfrom the lipid compartment by the exposure to heat in vivo.

If such mechanisms are operative, it argues well for the useof hyperthermia in combination with liposomes containing li-pophilic as well as polar drugs. This could be an importantconsideration as it has been shown that large amounts oflipophilic drugs can be incorporated into liposomes made froma variety of different lipids (12). For example, actinomycin Dwhich has a partition coefficient (octanol:water) of 91:1 had anefficiency of drug incorporation into liposomes of 28 to 66%,depending on drug concentration in the sonicating medium,whereas 1-/?-D-arabinofuranosylcytosine with a partition coef

ficient of 0.0012 only incorporated 0.5 to 1.2%. Juliano andStamp (13) have shown that nonpolar drugs, which can perturbthe phase transition behavior of synthetic phosphotidylcho-lines, are also maximally released from liposomes at the transition temperature of the constitutent lipids. Because many ofthe currently available cytotoxic drugs are lipophilic, a greaterdiversity of liposome drug combinations with heat could bepossible.

Using a drug similar to PDD, c/s-dichlorobiscyclopentylami-

neplatinum(ll), which has a very low solubility in both water andorganic solvents, Deliconstantinos ef al. (2) found that, uponliposome association, the drug was more readily incorporatedinto tumors of mice than was the free drug. The 15-fold increase

obtained in tumor drug concentration was ascribed to a slowerrate of liposome clearance from the circulation than of freedrug. The concentration in the liver, after administering thedrug associated in liposomes, was 3.5 times greater than thatobtained after free-drug treatment (2), which limits its value.

Since we routinely allowed the liposomes to stand overnightin the presence of free drug, the influence of this procedure ondrug distribution between small and large liposomes was studied (data not shown). If allowed to stand overnight prior toremoving free drug, 75% of the bound PDD radioactivity wasfound in the multilamellar fraction. If separated from free drugimmediately after sonication, the radioactivity distributed nearlyequally between small and large liposomes. The liposome-

associated radioactivity in both these experiments totaled approximately 15% of the initial radioactivity in the sonicatingsolution. The unexpectedly high proportion of large liposomesobtained immediately after sonication (1 hr) may be due tofusogenic properties of the PDD at the concentrations used inour studies. The overnight increase in the large liposomepopulation is in line with similar previous observations (26, 32).

As our results indicate that PDD is quite lipophilic, it is notsurprising that the blood clearance rate of liposomal PDD asseen in Chart 2 was only very slightly altered by local tumorhyperthermia, where the tumor transit time of the liposomes, atbest, should be in the order of seconds. Hyperthermia treatment did, however, have a clear effect on PDD uptake by theheated tumor. The increased uptake of drug from heatedvesicles is most likely the result of a number of factors, such asthe loss of the drug from the aqueous compartment of theliposome, a heat-induced increase in liposomal lipid uptake by

the tumor tissue, and, as indicated by the results in Chart 1,some release of bilayer-associated drug.

It is noteworthy that uptake of PDD radioactivity from smallliposomes relative to PDD administered as either free drug or

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Delivery of Liposome-associated FDD

in large liposomes was also enhanced in unheated tumors(Table 1). The greater time the small liposomes remain incirculation compared to free drug (Chart 2) could easily explainthe greater incorporation observed. Similarly, Dapergolas era/.(1) also obtained greater uptake in tumors when '"In-labeled

bleomycin was administered associated in small liposomescompared to drug given either in the free form or associatedwith large liposomes.

Our general conclusions from the current study with regardto the use of PDD-containing liposomes, alone or in combination with hyperthermia, are as follows: (a) FDD-associated small

liposomes have a significantly greater circulation time relativeto either free drug of PDD-containing large liposomes; (o) as a

result of the longer circulation time, uptake of radioactive PDDin the unheated tumor is higher after administration of smallliposomes; (c) heating the tumors after injection of small liposomes resulted in an additional increase in drug uptake by theheated tumor; (cO administration of PDD associated in liposomes markedly alters its distribution in other tissues, such asliver and spleen, particularly when administered associated inlarge liposomes; (e) association of PDD in liposomes withspecific T,, when combined in vivo with hyperthermia, delayedtumor growth; and (f) although hyperthermia enhanced the freePDD effect, a greater growth delay was obtained by the combination of liposomes and hyperthermia.

Accumulation of drug in the liver was only doubled when thePDD associated in small liposomes was administered, whileuptake by the kidney was essentially unchanged compared tofree drug. The failure to influence uptake by the latter organ isunfortunate as the major dose-limiting effect of PDD in humansis nephrotoxicity (1, 5, 10, 16). Nevertheless, relative to tumorconcentration, uptake in the kidney is reduced when the drugis administered associated in liposomes. While we are encouraged by the fact that specific release of PDD from liposomesby heat in the region of a tumor occurs, resulting in a greatertumor uptake of drug and longer growth delay, potential normaltissue (/.e., liver) toxicity problems need to be resolved beforeclinical application of this combined modality will be feasible.

ACKNOWLEDGMENTSWe are grateful to S. Engelmann-Brunner and H-J. Weber for help and advice

in various phases of this study. H. Röttgerand F. Wiertz for the tumor transplantation, P. Schmilz for the irradiation of PDD, K. Kasperek for the activationanalysis of PDD, and Dr. Peter Lelkes for critical reading of the manuscript.

REFERENCES

1. Dapergolas, G., Nerrunjun, E. D.. and Gregoriadis, G. Penetration of targetareas in the rat by liposome-associated bleomycin. glucose oxidase, andinsulin. FEBS (Fed. Eur. Biochem. Soc.) Lett., 63:235-239, 1976.

2. Deliconstantinos, G., Gregoriadis, G., Abel, G., Jones, M., and Robertson,D. Incorporation of c/s-dichlorobiscyclopentylamine platinum (II) in liposomes and its tissue distribution in tumor-bearing mice. Biochem. Sci.Trans., 5. 1326-1328, 1977.

3. Einhorn, L. H., and Donohue, J. c/s-Diamminedichloroplatinum, vinblastine,and bleomycin combination chemotherapy in disseminated testicular cancer.Ann. Intern. Med., 87: 293-298, 1977.

4. Fendler, J. H., and Romero, A. Liposomes as drug carriers. Life Sci., 201109-1120, 1977.

5. Gonzalez-Vitale, J. C., Hayes, D. M., Cvitkovic, E., and Sternberg, S. S. Therenal pathology in clinical trials of c/s-platinum (II) diamminedichloride.Cancer (Phila.), 39. 1362-1369. 1977.

6. Gregoriadis. G. Targeting of drugs. Nature (Lond.), 265. 407-411, 1977.7. Gregoriadis. G., Wills, E. J., Swain, C. P., and Tavill, A. S. Drug carrier

potential of liposomes in cancer chemotherapy. Lancet, 1313-1319, 1974.8. Haest. C. M. W., deGier, J., Van Es, G. A.. Verkleij, A. J., and van Deenen,

L. L. M. Fragility of the permeability barrier of Escherichia coli. Biochim.Biophys. Acta. 280. 43-53, 1972.

9. Hahn, G. M. Potential for therapy of drugs and hyperthermia. Cancer Res.,

39:2264-2268, 1979.

10. Hardaker, W. T., Stone, R. A., and McCoy. R. Platinum nephrotoxicity.Cancer (Phila.), 34: 1030-1038, 1974.

11. Juliano, R. L., and Stamp, D. The effect of particle size and charge on theclearance rates of liposomes and liposome encapsulated drugs. Biochem.Biophys. Res. Commun., 63: 651-658, 1975.

12. Juliano, R. L., and Stamp, D. Pharmacokinetics of liposome-encapsulatedanti-tumor drugs. Studies with vinblastine, actinomycin D. cytosine arabi-noside, and daunomycin. Biochem. Pharmacol., 27: 21-28, 1978.

13. Juliano, R. L., and Stamp. D. Interaction of drugs with lipid membranes:characteristics of liposomes containing polar or non-polar anti-tumor drugs.Biochim. Biophys. Acta, 586: 137-145, 1979.

14. Kimelberg, H. K., and Atchison, M. L. Effects of entrapment in liposomes onthe distribution, degradation, and effectiveness of methotrexate in vivo. Ann.N. Y. Acad. Sci., 308: 395-409, 1978.

15. Kimelberg, H. K., and Mayhew, E. Properties and biological effects ofliposomes and their uses in pharmacology and toxicology. CRC Crit. Rev.Toxicol., 6:25-79. 1978.

16. Madias, N. E., and Harrington. J. T. Platinum nephrotoxicity. Am. J. Med..65. 307-312, 1978.

17. Marmor, J. B. Interactions of hyperthermia and chemotherapy in animals.Cancer Res., 39: 2269-2276, 1979.

18. Mayhew, E., Papahadjopoulos, D., Rustum, Y. M., and Dave, C. Use ofliposomes for the enhancement of the cytotoxic effects of cytosine arabi-noside. Ann. N. Y. Acad. Sci., 308: 371-384, 1978.

19. Osieka. R., and Schmidt, C. G. c/s-Diamminedichloroplatinum (II), a newantineoplastic agent derived from the group of heavy metal complexes. Klin.Wochenschr., 57: 1249-1258, 1979.

20. Pagano, R. E., and Weinstein. J. N. Interactions of liposomes with mammaliancells. Annu. Rev. Biophys. Bioeng., 7: 435-468, 1978.

21. Papahadjopoulos. D., Jacobson, K., Nir, S., and Isac, T. Phase transition inphospholipid vesicles fluorescence polarization and permeability measurements concerning the effect of temperature and cholesterol. Biochim. Biophys. Acta, 311: 330-348, 1973.

22. Papahadjopoulos, D., Poste, G., Schaeffer, B. E., and Vail, W. J. Membranefusion and molecular segregation in phospholipid vesicles. Biochim. Biophys. Acta. 352: 10-28, 1974.

23. Rosenberg, B. Platinum coordination complexes in cancer chemotherapy.Naturwissenschaften. 60: 399-406. 1973.

24. Rosencweig. M., von Hoff, D. D.. Slavik, M., and Muggia, F. M. c/s-Diamminedichloroplatinum (II) a new anticancer drug. Ann. Intern. Med., 86:308-312, 1977.

25. Steel, G. G., and Peckham, M. J. Explantable mechanisms in combinedradiotherapy-chemotherapy: the concept of additivity. Int. J. Radiât.Oncol.Biol. Phys.. 5: 86-91, 1979.

26. Suurkuusk, J., Lentz, B. R., Barenholz, Y., Biltonen, R. L., and Thompson,T. E. A calorimetrie and fluorescent probe study of the gel-liquid crystallinephase transition of small single lamellar dipalmitoyl phosphatidylcholinevesicles. Biochemistry, 15: 1393-1401, 1976.

27. Tsong, T. Y. Transport of 9-anilino-1-naphthalene-sulfonate as a probe ofthe effect of cholesterol on the phospholipid bilayer structures. Biochemistry,14: 5415-5417, 1975.

28. Tyrell, D. A., Heath, T. D.. Colley. C. M., and Ryman, B. E. New aspects ofliposomes. Biochim. Biophys. Acta, 457: 259-302, 1976.

29. Weber, H. J. In vivo Untersuchungen am Experimentaltumor Sarkom-180überdie Therapieaffizienz von Gammastraklen und Neutronen in Verbindungmit Hyperthermie und Misonidazol (doctoral dissertation). Düsseldorf: University of Düesseldorf, 1981.

30. Weber, H. J., Porschen, W., Dietzel. F., Mühlensiepen, H., and Feinendegen,L. E. In vivo analysis of the influence of combined hyperthermia and gammairradiation on euoxic and hypoxic tumor cells. In: C. Streffer, D. van Beunin-gen, E. Dietzel, E. Rottinger, J. E. Robinson, E. Scherer, S. Seeber, and K.-R. Trott (eds.). Cancer Therapy by Hyperthermia and Radiation, pp. 276-277. Baltimore: Urban & Schwarzenberg, 1978.

31. Weinstein, J. H., Magin, R. L., Yatvin, M. B., and Zaharko, D. S. Liposomesand local hyperthermia: selective delivery of methotrexate to heated tumors.Science (Wash. D. C.), 204: 188-191, 1979.

32. Weissmann, G., Bloomgarden, D., Kaplan, R., Cohnen, C., Hoffstein, S.,Collins, T., Gotlieb, A., and Nagle, D. A general method for the introductionof enzymes, by means of immunoglobulin-coated liposomes, into liposomesof deficient cells. Proc. Nati. Acad. Sei. U. S. A., 72: 88-92. 1975.

33. Wolf. W.. Mamaka, R. C., and Ingalls. R. B. Radiopharmaceuticals in clinicalpharmacology '95ctâ„¢-c/s-diamminedichloroplatinum (II). New developments

in radiopharmaceuticals and labelled compounds. Proceedings of the International Atomic Energy Agency, Symposium Copenhagen, March 26-30,1973.

34. Yatvin, M. B., Weinstein, J. N.. Dennis. W. H.. and Blumenthal, R. Use ofhyperthermia to promote local release of drugs from liposomes. In: W. L.Caldwell and R. E. Durand (eds.). Clinical Prospects for Hypoxic CellSensitizers and Hyperthermia. vol. 2, pp. 105-112. Madison. Wis.: Wisconsin Press, 1977.

35. Yatvin, M. B., Weinstein, J. N., Dennis, W. H., and Blumenthal, R. Design ofliposomes for enhanced local release of drugs by hyperthermia. Science(Wash. D. C.), 202: 1290-1293, 1978.

MAY 1981 1607

on March 13, 2021. © 1981 American Association for Cancer Research.cancerres.aacrjournals.org Downloaded from

1981;41:1602-1607. Cancer Res   M. B. Yatvin, H. Mühlensiepen, W. Porschen, et al.   Tumor Drug Uptake and Growth-Dichlorodiammineplatinum(II) by Heat and Its Influence on

cisSelective Delivery of Liposome-associated

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