BASICSCIENCES

Although liposomes have been investigated in detailfor applications such as drug transport and cell-membrane studies, their potential in nuclear medicine has notbeen fully established. Several studies have investigatedthe biodistributions of gamma-emitting radionuclidescontained in either the aqueous or the lipid liposomephase(1—15).

All of these previous studies have used liposomescomposed mainly of phosphatidylcholine (lecithin) witha small amount ofcholesterol to improve stability. Sincelecithin is an uncharged zwitter-ion at neutral pH, smallamounts of charged lipids, such as dicetyl phosphate,

Received Sept. I 1, 1979; revision accepted Feb. 12, 1980.For reprints contact: D. J. Hnatowich, PhD, Dept. of Nuclear

Medicine, Univ. of Massachusetts Medical Ctr., Worcester, MA01605.

have been added to provide a surface charge. Studieswith these charged liposomes have demonstrated a smallbut significant effect of charge on biodistribution (10,12-14).

A majorattractionof liposomesisthealmostunlimited variation possible in the composition of the liposomemembrane. Since surface charge has been shown to bea determinant of biodistribution, it is reasonable thatincreasing liposome charge through the use of novel lipidcompositions may increase the effect on biodistribution.This may be best accomplished not by adding smallamounts of charged lipids to lecithin, but rather by replacing lecithin with a charged lipid capable of formingliposomes.

We have prepared liposomes composed primarily ofcardiolipin, a molecule similar in structure to lecithin butpossessing a double negative charge at neutral pH, as

662 THE JOURNAL OF NUCLEAR MEDICINE

RADIOCHEMISTRY AND RADIOPHARMACEUTICALS

Investigationsof a New, Highly Negative Liposome with ImprovedBiodistribution

for Imaging

D. J. Hnatowich and B. Clancy

University of MassachusettsMedical Center, Worcester, Massachusetts

An aftractivefeatureof liposomesisthe wIde rangeof lipidcompositionthat canleadto liposomeformation,coupledwiththe observationthat liposomebiodistributIonmay be alteredby varyinglipidcompositIon.For Instance,addingchargedlipida to neutral iecfthlnwill after the blodistrlbutionof the resuftingcharged liposomes. We have prepared highly negatIve liposomes by replacing lecfthln wfthnegativelychargedcardlolipin.Theliposomeshavebeenlabeledin the lipidphasewith Ga-Si and Tc-99m oxine and their propertiesevaluated.The expected highnegativecharge of the resuftingliposomeswas confirmedby an Ion-exchangechromatographictechnique.Usingpaperchromatography,thestabilityof the labelwas determi,@ during incubationin saline and serum. Finally, biodistributlonswere determinedat 2 hr in mice, andthe resultscomparedwiththosefor negativeIecfthin llposomes.Accumulatedactivfties in lIver and spleen were reduced byfactorsof five and 20, respectively,over lecfthinilposomes.Sincepreferentialaccumulationof actIvity in these organsconstftutesthe biggestlimitationto the useof lecithin ilposomes,cardiolipinliposomesmay proveto be more usefulcarriersof radioactlvftyin imagingapplicatIons.More importantly,however, these resultsillustrate the value of studying novel liposome types as potential radiopharmaceuticals.

J Nuci Med 21: 662—669,1980

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BASIC SCIENCESRADIOCHEMISTRYAND RADIOPHARMACEUTICALS

shown in Fig. I . The liposomes have been labeled in thelipid phase with both Ga-67 oxine and Tc-99m oxine.The relative surface charges of these liposomes have beenmeasured by an anion-exchange column technique. Inaddition, the stability of the labeled liposomes has beendetermined in both saline and serum environments. Finally, biodistributions of the Ga-67 liposomes has beendetermined in normal mice at 2 hr and the results compared with those for negative lecithin liposomes.

MATERIALS AND METHODS

Liposome preparation. Liposomes were prepared bystandard methods. Neutral liposomes were composed oflecithin (L@-phosphatidyl choline dipalmitoyl) andcholesterol (molar ratio 7 : 1); conventional negative Iiposomes were composed of lecithin, dicetyl phosphate,and cholesterol (molar ratio 7 : 2 : I); and positive liposomes were composed of lecithin, stearylamine, andcholesterol (molar ratio 7 : 3 : 2). Cardiolipin liposomeswere composed of cardiolipin (diphosphatidyl glycerol)and cholesterol (molar ratio 7 : 2). Stearylamine,* cardiolipin,t and the remaining chemicalst were obtainedfrom commercial sources and were used without furtherpurification.

Gallium-67 was obtained as the citrate radiopharmaceutical. Benzyl alcohol present in the radiopharmaceutical extracts into chloroform, along with the oxinecomplex, and is not removed during rotary evaporationin the preparation of liposomes. Therefore, it was firstremoved by extraction into chloroform. The pH of theGa-67 citrate solution was then adjusted to about 5 bythe addition of 2 ml of 0.05 M sodium acetate buffer, pH

5.6, to the 4-mi aqueous phase. The oxine complex wasthen formed by the addition of I00 @lof an ethanol solution of oxine containing 2 mg/mI of oxine.@The Ga-67oxine complex was then extracted into two 2-mi volumesofchloroform. The extraction was usually repeated with50 @lof oxine solution and the chloroform extractscombined. Normally about 50-80% of the Ga-67 activitywas recovered.

Technetium-99m was obtained as pertechnetate froma generator. The oxine complex was prepared by addingtogether 1 ml of a fresh, nitrogen-purged solution contaming so zg of SnCi2.2H20 and 100 @zlof the aboveoxine solution. The pH was then adjusted to between 8-9with dilute NaOH, and I -3 mCi of pertechnetate solution added. The oxine complex was extracted with 2-3ml ofchioroform. The recovery ranged between 70 and90%.

Approximately 10 mg of the appropriate lipids weredissolved in 4-6 ml of chloroform in a round-bottomflask and mixed with either the Ga-67- or the Tc99m-oxine chloroform solution. The flask was thenconnected to a rotary evaporator and the contentsevaporated in 30—45mm under reduced nitrogen pressure while the flask was maintained at 60°Cin a waterbath. Following evaporation, I ml of either 0.9% NaC1(pH 7.5) or 0.05 M PBS (pH 7.3) was added to the flaskand the lipids coating the walls dispersed with a magneticstirring bar. The flask was then placed in a bath sonicator'1 for I —10 mm in the case of cardiolipin iiposomesand 60 mm for all other liposomes. During sonication,the liposomes were kept at or below room temperatureby periodic addition of ice to the bath. Labeled liposomeswere separated from free activity by chromatography

0II

CH3-(CH2)@—C—0-CH2

01 ___II LECITHINCH3—(CH2@mC—0-CHII

CH2—0 F@—CH2—CH2—N(CH3)3OG

DICETYL PHOSPHATE

0CH3—(CH2)30-CH2—0—I@—OH

oeFIG. 1. Structtwal formulae of three compounds pertaIning to this research.

0 0II II

CH3—(CH2)@—C—O--CH2 CH2OC(CH2)nCH30 I CARDIOLIPIN I 0II I I ii

CH3(CH2)mCOCH 0 OH@ 0 CH0C(CH2)mCH3I II I II ICH2—OP—CH2—CH—CH2—O 2

00 oe

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IINATOWICII A\I) (lANCY

on a I .5- X 20-cm column of Sephadex G-50 using 0.05M PBS as ciuant. The milky suspension eluting near thevoid volume contained the radiolabeled liposome fractions. The labeling efficiency was determined bycounting the liposome-containing fractions either in aNaI(Tl) well counter or in an ionization dose calibratorand comparing with a standard of the unfractionatedsuspension. Each preparation was inspected using a lightmicroscope to ensure that the liposomes were uniformin size and free of aggregates.

Surface-chargemeasurements.Ion-exchangechromatography was investigated for the purpose of measuring reiative liposome surface charge. Neutral, nega

tive, and positive lecithin liposomes labeled with Ga-67were applied to different column lengths of AG I X 4anion-exchange resin and AG-SO X 8 cation-exchangeresin. The effects ofeluant composition, resin mesh size,column flow rate, etc., on the elution of activity fromthese columns were determined for each of these liposome types. On the basis of these measurements, ananion-exchange column was constructed to determinethe relative charge of negatively charged liposomes.

Stability studies. Since cardiolipin-cholesterol liposomes have not been studied previously, a method wasrequired for estimating the relative stability of the labelduring incubation in saline and serum. Paper chromatography, using Whatman No. I paper and saline eluant,pH 7, was used to determine the fraction of activitybound to liposomes. Negative lecithin liposomes andcardiolipin liposomes labeled with Ga-67 or Tc-99mwere incubated in 1 : I dilution ofsaline or fresh humanserum with frequent agitation for up to 2 hr at both 20and 37°.Samples were taken for paper chromatographyat 5 mm and I and 2 hr. In all cases, after spotting, thepapers were not permitted to dry before chromatography. This stability assay not only determined the fractionof activity still bound to liposomes, but also determinedthe chemical form of the released activity.

Biodistributions. In this study mice were killed 2 hrafter injection, to ensure that liposome degradation, withrelease of label to the circulation, does not contributesignificantly to the distribution. Cardiolipin liposomepreparations were used for biodistribution determinations immediately after swelling and gel chromatography. Lecithin liposomes were occasionally stored overnight in a refrigerator before use, since stability studiesdemonstrated that only slight dissociation occurredunder these conditions.

Approximately I mg of lipids in 0. 1-0.2 ml of salinecontaining about 5 @.zCiof activity was administered bytail vein to each CD-I male mouse1. Following etherization of each animal at 2 hr. a blood sample was obtamed by cardiac puncture and tissue samples were removed for weighing and subsequent counting in aNal(Tl) well counter. Samples of bone were obtainedfrom the intact femur. Results were obtained for Ga-67

negative lecithin and cardiolipin liposomes, and forGa-67 oxine itself; results are expressed as percentageinjected dose per gram wet weight, normalized to a 25-gmouse. In the case of Ga-67 oxine, the activity was administered in 50% ethanol.

To ensure that the distributions observed were notinfluenced significantly by unidentified variables of thepreparations, two preparations of each liposome typewere studied and the results are reported separately.

RESULTS

The average Ga-67 labeling efficiencies were I 5, 80,and 60% for neutral, negative, and positive liposomes,respectively, and 10% for cardiolipin liposomes. TheTc-99m labeling efficiency for cardiolipin liposomes wassimilar. Although all lecithin liposome types appeared

to be uniform in size on the basis of light microscopy, theparticle-size distribution of the cardiolipin liposomesappeared to be smaller. This was confirmed by centrifugation measurements, in which 20% of the activity ina cardiolipin liposome preparation would typically be

centrifuged under specific conditions, compared with45% for lecithin liposome preparations. In order to prepare cardiolipin liposomes with size distributions similarto those of the other liposome types, sonication time wasreduced to between I and 10 mm. The same fractionnumbers from G-50 chromatography were collected foruse.

An electron-microscopic examination of a cardiolipinliposome preparation was achieved by mixing 1 drop ofan unfractionated liposome preparation (swelled in

FIG.2.Electronmicrographofacardlolipinliposome.

L@_@ O.IpM

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BASIC SCIENCESRADIOCHEMISTRYAND RADIOPHARMACEUTICALS

distilled water rather than saline) with 1 drop of a 2%phosphotungstate solution and allowing themixture tostand for 20 mm. It was then evaporated to dryness ona copper grid and placed in an electron microscope.Figure 2 is an electron micrograph obtained in thismanner; it clearly shows the multilaminar structure ofthe liposome.

As mentioned previously, the relative liposome surfacecharge was determined by ion-exchange chromatography. Neutral, negative, and positive lecithin liposomeslabeled with Ga-67 were applied to columns of anionand cation-exchange resins. A 50—100-mesh size wasfound to be suitable and was chosen to reduce the likelihood of physical trapping. Figures 3 and 4 illustrate thebehavior of the liposomes on I .5- X 20-cm columns ata flow rate of 20 sec/ml. In Fig. 3, results of passingneutral, negative, and positive liposomes through theanion-exchange AG I column are shown; negative Iiposomes are eluted to a negligible degree relative toneutral and positive liposomes, The reverse is true on thecation exchanger AG 50, as shown in Fig. 4. Surprisingly, it is not the position of the peak that is affected byliposome charge, but rather its magnitude. A possibleexplanation assumes that liposomes of appropriatecharge have strong affinity for the resin's binding sites,and these sites are easily saturated. Activity would thenelute from the column only if the charge and numbersof binding sites are appropriate. The fact that neutralliposomes and liposomes with the same charge as theresin are not eluted completely is possibly due to thezwitter-ion nature of lecithin. In support of these suggestions, we have observed that the degree of elution forall liposome types may be increased by shortening thecolumns or by increasing the flow rate.

Additional evidence that these phenomena are due toeffects of liposome charge was obtained by measuringthe degree of elution of negative liposomes from an AG

FIG.3.Percentagesofneutral,negative,and positive lecithin Ilposomes per fractioneluting from anion-exchange column.

1 column with eluants at different pH . The results of thisstudy are shown in Fig. 5. The degree ofelution is constant at neutral and basic pH but increases with decreasing pH below about 4. This behavior is easily explained, since dicetyl phosphate—the compound thatprovides the negative charge on these liposomes—hasa PKa between I and 2 (16). Consequently as the pH ofthe eluant approaches 2, the liposome will begin to loseits negative charge and be eluted as neutral. This studywas performed both with distilled water and with 0.9%NaCl eluant. As expected, the presence of NaC1 did notaffect the shape of the curve, but did increase the degreeof elution at each pH value. This effect is due to thecompetition between chloride ions and liposomes for theresin's binding sites.

To quantitate the surface charge of negative liposomes, a I .5- X 5-cm AG I column was prepared andused with distilled-water eluants, pH 7, and at the sameflow rate. The column was calibrated with radiolabeledneutral liposomes (lecithin and cholesterol, molar ratio7 : 2), negative liposomes (lecithin, dicetyl phosphate, andcholesterol, molar ratio 7 : 2 : 1) and “half-negative―liposomes (lecithin, dicetyl phosphate, and cholesterol,molar ratio 7 : 1 : 1). The percentage of activity elutednear the void volume was determined for each of thesepreparations. The results appear in Fig. 6; each datumis the average of at least three determinations, usuallyon more than one preparation. The bars show the rangesof measurements. The charge density of the liposomes,based on the molar percentage of the dicetyl phosphate(and assuming a charge of 1.5 per molecule at neutralpH), is plottedon the abscissarelativeto that of negativelecithin liposomes. The curve shows the expected dependency of percent elution on liposome charge. Cardiolipin liposomes with a charge density of 2.16 displayeda 10 ±5% recovery under these conditions, confirmingthe expected high negative charge of these liposomes.

ANIONIC EXCHANGE

21 0 PositiveU Negative

S NeutralC0

U0

U-

@ll

FRACTION

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HNATOWICH AND CLANCY

CATIONIC EXCHANGE

0 Positive. NegativeB Neutral

2@

C0U0U-

>

U

•0a,

UiFIG.4.Percentagesofneutral,negative,and positive lecithin liposomes per fractioneluting from cation-exchange column.

15

FRACTION

Paper chromatography, on Whatman No. 1paper andsaline pH 7 eluants, was found to be suitable for measuring the loss of label from Ga-67 and Tc-99m liposomes. Being insoluble, activity bound to liposomes doesnot migrate from the origin in this system. Activitypresent in water-soluble forms travels from the origin,and may therefore be distinguished from activity presenton liposomes. However, both Ga-67 and Tc-99m mayexist in aqueous solution as insoluble and colloidal oxidesor hydroxides, and their presence will contribute to theorigin activity peaks. Since each liposome preparationwas purified by gel chromatography, however, theseimpurities will be absent; both Ga(OH)3 and colloidalTc-99m (probably Tc02) were found either to bind irreversibly to G-50 columns or to elute slowly and notalong with the liposomes. Furthermore, preparations ofthese insoluble forms of Ga-67 and Tc-99m (as well asthe oxine complexes) were incubated in serum at 37°inthe usual manner and chromatographed. The percentage

I00

90

80

70

60

50

40

30

20

10

of activity at the origin in these chromatograms variedbetween 1 and 8% over the 2-hr incubation.

Figure 7 shows typical radiochromatograms obtainedfor Ga-67 lecithin and cardiolipin liposomes for preparations incubated in saline and serum at two temperatures. In the case of Ga-67, the chemical form of thesoluble activity released from the liposomes is thoughtto be a colloidal Ga oxine displaying an Rf value in thissystem of about 0.09 (soluble Ga-67 oxine has an R@of0.42). In the case of Tc-99m, the principal solublechemical form following incubation in saline is pertechnetate (R@0.70), whereas in serum it is the oxinecomplex (R@0.55).

The percentage of activity bound to liposomes at 5 mmin saline is a measure of the radiochemical purity of the

00

90

80@

>.

U

‘0a)

w

. 09% N0CI

0 D,st Woter

7C

6C

50

40

3C

20

IC

- .0 2.0

Liposome Charge Density

FIG.6.EffectofIlposomechargeonpercentageofliposomeselutingInvoIdvolumefromananIon-exchangecolumn.Liposomechargedensityrelativetothatofconventionalnegativellposomesisplottedon abscissa.

a,Va

w

@, I 2 3 4 5 6 7 8 9 0pH

FIG.5.PercentagesofnegativelecIthinIlposomeselutingInvoIdvolume from an anIon-exchange column, as functIons of eluantacIdIty for both 0.9% salIne and dIstIlled water.

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SalineSalineSerumSerum20°C37°C20°C37°C

2Hj@@J@

:ii@@A@

BASIC SCIENCESRADIOCHEMISTRYAND RADIOPHARMACEUTICALS

67GaLecithinLiposomes 67GoCordiohpsnLiposomesTABLE1. PERCENTDISSOCIAT1ONOF Ga-67

AND Tc-99m CARDIOLIPINAND LECITHINLIPOSOMESIN SALINEAND SERUM

‘)Mn 5 M@n

Ga-67 lecithinliposomes5mm043832lIT31029232hr812 29

Ga-67cardlolipinIlposomes255mm1103139ltw17527452hr0

11 39Tc-99m lecithinliposomes485mm0

015561I@184129482hr2011 39

Tc-99m cardiolipinliposomes795mm000531I@25

436792hr2464 3979

Hour

Origin • SolventFront

SosoentFront

Orgin

FIG. 7. Radiochromatogramsobtainedat 5 mm and 1 and 2 hr forGa-67-labeled lecIthin and cardlolipin Ilposomes Incubated in salineandserumat 37°C.ActIvityboundto IlposomesremaInsat origin;released soluble activity migrates with solvent.

liposome preparations. Typically, for negative lecithinliposomes about 90% of Ga-67 activity is liposome boundimmediately following G-50 separation, whereas forcardiolipin liposomes this value is about 60%. Onstanding at room temperature—and especially at elevated temperatures—these values decrease. This is apparent from Table 1, which lists the percent dissociation,as determined by chromatographic assay, at 5 mm and1 and 2 hr, for I : I dilutions of the preparations in salineand in serum. Little dissociation of either liposome typeoccurs in saline at either temperature, whereas considerably more loss of label occurs in serum at both temperatures. The results obtained for Tc-99m-labeled Iiposomes, also presented in the table, show these to be

much less stable to the release of activity than are theGa-67 liposomes.

Biodistributions were obtained only for the Ga-67-labeled liposomes, because of their greater stability. Theresults obtained for two preparations each of negativelecithin and cardiolipin liposomes are presented in Table2 along with that for Ga-67 oxine itself. Clearly thebiodistribution of the label has been significantly alteredby changing the carrier liposome from negative lecithinliposomes to the highly negative cardiolipin liposomes.In particular, accumulation in liver has decreased by afactor of about five, while that in the spleen has decreased by a factor ofabout 20. In addition, blood, lung,and kidney levels have also decreased, whereas uptakein bone and muscle has increased.

By consideringthe relativeweights of each organ and

TABLE2. BIODISTRIBUTIONSAT 2 HR IN NORMALMICE FOR Ga-67 OXINEAND FOR TWOPREPARATIONSEACHOF Ga-67 CARDIOLIPINAND NEGATIVELECITHINLIPOSOMES

Ga-67 cardlolloin Iloosomes Ga-67 lecithin Iloosomes Ga-67oxine

Blood3.4 ±1.02.9± 0.88.5 ± 1.36.9± 2.99.1±3.2Brain—0.30±0.100.20 ± 0.010.22± 0.120.21±0.06Liver6.9

±3.15.1± 0.721 ± 524± 126.0±1.1Spleen3.6±1.75.7±1.970±1998±783.9±1.1Lung2.2

±0.63.3± 0.77.1 ± 2.28.0± 3.818±19Kidney2.0±0.51.7±0.43.6± 0.34.6±1.95.00.6Heart2.1

±0.81.9± 0.72.2 ± 0.22.2± 0.84.1±0.9Bone9.9@3.17.3±2.04.0± 0.88.5± 4.04.9±0.8Muscle2.8

±2.51.2± 0.60.80 ± 0.171.0± 0.51.5± 0.4

. Results expressed as % injecteddose/g normalized to unftbody weight with 1 s.d.of the mean. Each datum Isthe average

of five animals.

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HNATOWICH AND CLANCY

tissue (17), we estimate that some 60% ± 14% of the

injected activity was accounted for in the case of the micestudies with Ga cardiolipin liposomes in contrast to 85%:1: 12% for the lecithin liposomes. This difference, ifsignificant, may indicate that the cardiolipin liposomesare cleared more rapidly into gut and/or urine than arethe lecithin liposomes.

DISCUSSION

Perhaps the most significant aspect of liposomes is thealmost infinite variety of lipid composition that can lead

to liposome formation. Of special interest to applicationsin nuclear medicine is the possibility that certain of thesevariations will effect biodistributions in a useful manner.One property of liposomes that may be readily alteredis surface charge. Previous work in this area has demonstrated an effect of charge on biodistribution, but allof these previous studies have apparently used liposomescomposed primarily of lecithin to which small admixtures ofcharged lipids have been added. Liposomesof much higher charge may be prepared by replacinglecithin with phospholipids that are themselves charged.For example, in this study we have used cardiolipin toprovide highly negative liposomes; other phospholipidsthat may be suitable are phosphatidylserine (18),phosphatidylglycerol (/9), and phosphatidic acid.Similarly, highly positive liposomes may be preparedfrom phosphatidylglycerol lysine (19). Cholesterol hasbeen reported to stabilize artificial lipid vesicles (11), andmay be an important addition to these liposomes.

In a study of this nature, the properties of the newliposome type must be carefully evaluated. Obviously,an effort must be made to ensure that liposome formation has actually occurred and that for comparisonpurposes the size distribution is similar to that of conventional liposomes. It must be possible to label the Iiposomes to good yield and to demonstrate that the labeled liposome is stable in serum environments at 37 °C.If the object is to prepare charged liposomes, somemethod must be available to measure this charge. Theion-exchange assay used in this work is simple and provides reproducible results. An electrophoretic methodof measuring relative liposome charge has also been used(19). It would be particularly interesting to measure thecharge following incubation in serum environments,since the presence in serum of ions, such as Ca2+, mayaffect liposome charge in vivo by binding to the liposomesurface (20). Finally, it is particularly important to ensure that duplicate preparations of each liposome typeprovide reproducible results.

The use of the paper chromatographic assay haspermitted the determination of the fraction of activitybound to liposomes in suspension of these particles. Ofparticular interest is the observation that, immediately

following G-50 chromatography, appreciable activityis present in a soluble chemical form, probably as theoxine complex. Since purification on Sephadex columnswill remove these low-molecular-weight species, theirpresence following chromatography must indicate thatrapid re-equilibration has occurred. Likewise, we haveshown that exhaustive equilibrium dialysis is only partially successful in removing these soluble species.Hwang (2! ) has shown that In-I I I oxine diffuses rapidlythrough the lipid bilayers of liposomes and exchangesreadily from one liposome to another. Because of itschemical similarity to indium, Ga-67 oxine may be expected to behave in a similar manner. Results from thisstudy indicate that the oxine complex of Tc-99m is evenmore prone to diffuse from lipid bilayers. These resultsare similar to those we have obtained in a separate studyof leukocytes labeled with Tc-99m oxine; they werefound to release their label much more readily thanleukocytes labeled with Ga-67 oxine.

An important result of this study of cardiolipin liposomes is the decreased accumulation of activity in liverand spleen relative to that observed in all previous studieswith lecithin liposomes. This observation is particularlynoteworthy, since the principal limitation of liposomeuse in nuclear medicine is the preferential localizationof activity in these organs. Although accumulation inliver and spleen can be reduced, often slightly, throughthe use ofsmall liposomes (8, 13, 22), subcutaneous andintraperitoneal administration (6), use of carrier stableliposomes (7, 8), incorporation of specific proteins (5),and novel methods of liposome preparation (9), thesemethods are not practical in all cases.

As previously mentioned, light microscopy and centrifugation measurements showed the size distributionof the cardiolipin liposomes to be smaller than that of thelecithin liposomes. This was so despite a much shortersonication time. Some decrease in liver and spleen accumulation with decreasing liposome size is expected,but the magnitude of this effect is far smaller than thatobserved in this work, since the reduction is by a factorof 2-4 in spleen uptake and 2 or less in liver uptake (8,13, 21). An increase in liver uptake with decreasingliposome size has been reported (5).

The labeling efficiency achieved in this work of about10% for the cardiolipin liposomes is a limitation thatmust be dealt with before imaging studies can be performed with this agent. Furthermore, a method of Iabeling liposomes of this and other types after theirpreparation, rather than during preparation as was thecase in this work would be highly desirable. A methodof labeling preformed lecithin liposomes with Tc-99mhas been reported (12), however, we were unable to forma stable label using this method. A promising approach,developed in our laboratory, is the incorporation of alipophilic compound containing a lipophobic chelatinggroup.

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FOOTNOTES

a Pfaltz and Bauer, Inc., Stamford, CT.

t Grand Island Biological Co., Grand Island, NY.

ISigma Chemical Co.,St.Louis,MO.

I Bronsonic Model 10-002,25 watts.

, Charles River Breeding Laboratories, Wilmington, MA.

ACKNOWLEDMENTS

The authors express their appreciation to S. Kulprathipanja, T. 0.Connell, and S. Y. Kopiwada for their assistance in the initial phasesof this work, and to J. Underwoodfor performing the electronmicroscopy. This work was supported in part by BRS Grant No.RRO57 I 2-07.

REFERENCES

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2. GREGORIADISG. RYMAN BE: Lysosomal Localization off3-fructofuranosidase-containing liposomes injected into rats.Biochemf 129:123-133,1972

3. GREGORIADIs G, RYMAN BE: Lysosomal localization of19-fructofuranosidase-containing liposomes injected into rats.Biochemf 129:123-133,1972

4. JULIANORL, STAMPD:Theeffectof particlesizeandcharge on the clearance rates of liposomes and liposome encapsulated drugs. Biochem Biophys Res Commun 63:651-658, 1975

5. DUNNICK JK, MCDOUGALL IR, ARAGON S, et al: Vesicleinteractions with polyamino acids and antibody: In vitro andinvivostudies.J NucIMed 16:483—487,1975

6. MCDOUGALL IR, DUNNICK JK, G0RIS ML, et al: Lii vivodistribution of vesicles loaded with radiopharmaceuticles: Astudy of different routes of administration. J NucI Med 16:488-491,1975

7. CARIDE Vi, TAYLOR W, CRAMERJA, et al: Evaluation ofliposome-entrappedradioactivetracersasscanningagents.Part I: Organ distribution ofliposome [@mTc@DTPAlin mice.JNucIMed 17:1067-1072,1976

8. GREGORIADIS G, NEERUNJUN DE, HUNT R: Fate ofliposome-associated agent injected into normal tumourbearing rodents.Attempts to improve localization in tumourtissues.LifeSci21:357-370,1977

9. DUNNICKJK,KRISsJP:Studiesofradiopharmaceuticalenclosing lipid-protein vesicles formed from native plasmacomponents.JNuclMed18:183-186,1977

Jo. CARIDEVJ,ZARETBL:Liposomeaccumulationinregionsof experimentalmyocardialinfarction.Science 198: 735-738,1977

Ii. DUNNICK JK, BADGER RS, TAKEDA Y, et al: Vesicle interactions with antibodyand peptide hormone:Roleof vesiclecomposition. J NucI Med I7: 1073- 1076, 1976

12. RICHARDSON Vi, JEYASINGH K, JEWKES RF, Ct al:Properties of99mlc technetium-labeled liposomes in normaland tumour-bearing rats. Biochem Soc Trans 5: 290-291,I977

13. RICHARDSONVJ, JEYASINGHK, JEWKESRF,etal: Possible tumor localization ofTc-99m-labeled liposomes: Effectsof lipid composition,charge, and liposomesize. J Nuci Med19:1049-1054,1978

/4. ESPINOLALG,BEAUCAIREJ,GOTTSCHALKA,etal:Radiolabeled liposomes as metabolic and scanning tracers inmice. II. In-I I I oxine compared with Tc-99m DPTA, entrapped in multilamellar lipid vesicles. J Nuci Med 20:434-444, 1979

15. HINKLE GH, BORNGS, KESSLERKy, et al: Preferentiallocalization of radiolabeled liposomesin liver.J PharmacolSd 67: 795-798, 1978

16. LEHNINGERAL: Biochemistry.New York, Worth Publishers, Inc., Second Ed , 1975, p 290

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