Analysis of the Fate of Systemically Administered ...Analysis of the Fate of Systemically...

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[CANCER RESEARCH 42. 1412-1422, April 1982] 0008-5472/82/0042-0000$02.00 Analysis of the Fate of Systemically Administered Liposomes and Implications for Their Use in Drug Delivery1 George Poste,2 Corazón Bucana, Avraham Raz, Peter Bugelski, Richard Kirsh, and Isaiah J. Fidler Smith Kline and French Laboratories. Philadelphia, Pennsylvania 19101 [G. P., P. B., R. K.¡;Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 [G. P.]; and Cancer Metastasis and Treatment Laboratory, National Cancer Institute-Frederick Cancer Research Facility, Frederick, Maryland 21701 ¡C.B., A. R., I. J. F.] ABSTRACT Functional and ultrastructural studies of liposomes injected i.v. into inbred C57BL/6N mice were performed to determine whether free liposomes can traverse capillaries. In the liver and spleen, organs with discontinuous (sinusoidal) capillaries, ul- trastructural and cell fractionation studies revealed that small (300- to 800-Â diameter), sonicated, unilamellar liposomes were more efficient in penetrating liver sinusoids to interact with hepatocytes than were large (0.5- to 10-jum) multilamellar liposomes. Ultrastructural studies of the behavior of liposomes in the continuous capillaries of the lungs revealed that circu lating phagocytic cells engulf the liposomes in the capillaries. Transcapillary migration of free liposomes was not observed. We conclude that free liposomes are unable to extravasate to reach the alveoli for subsequent engulfment by alveolar mac rophages. Instead, liposomes in the lung capillaries are en gulfed by circulating blood phagocytes which subsequently migrate to the alveoli to become alveolar macrophages. Exper iments on the recruitment of blood monocytes into the lungs subjected to whole- or partial-body X-radiation confirmed that transfer of i.v.-injected liposomes to the alveolar compartment was mediated by blood monocytes. The inability of liposomes to escape from continuous capillaries and their rapid uptake by circulating and fixed phagocytic cells calls into question the feasibility of using liposomes to "target" drugs to cells in extravascular tissues. INTRODUCTION The ability to selectively "target" cytotoxic drugs to tumor cells in vivo has been a long-cherished goal in cancer therapy. In the last few years, liposomes have attracted considerable interest as potential candidates for accomplishing this task (for reviews, see Refs. 13 and 19). Recent in vitro studies have demonstrated that liposomes bearing cell-specific antibodies react with antigen-bearing target cells with much greater effi ciency than do liposomes without surface antibodies (14, 15, 22). Although encouraging, similar targeting to specific cells in vivo has not been successful. Clearly, many obstacles must be overcome to allow liposomes to interact with the desired target ' Research sponsored by the National Cancer Institute, Department of Health and Human Services, under Contract N01-CO-75380 with Litton Bionetics, Inc. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the United States Government. 2 Recipient of USPHS Grants CA18260 and CA30192. To whom requests for reprints should be addressed, at P. O. Box 7929, Smith Kline and French Laboratories, Philadelphia, Pa. 19101. Received September 22, 1981; accepted January 4. 1982. cell. For example, to treat solid tumors or their métastasesthat proliferate in the extravascular compartments of major organs, liposomes containing drugs would be injected into the circula tion and thus would have to traverse capillaries to reach the tumor cells (4). If transcapillary passage of intact liposomes cannot occur, then the effort of constructing liposomes with sophisticated ligands that "recognize" tumor cells would be futile because the mechanical barrier imposed by the microcir culation would prevent liposomes from reaching the intended target cell. Information on the transcapillary transport capabil ities of liposomes is thus crucial for critical evaluation of the potential use of liposomes as carriers of cytotoxic drugs in cancer therapy. In this study, we have examined the ability of liposomes of differing size to cross anatomically different classes of capil laries. Our results indicate that limited transcapillary transport of liposomes following i.v. injection occurs in open sinusoidal capillaries (liver) but does not occur in organs with continuous capillaries (lung). Moreover, we demonstrated the efficient uptake of liposomes by circulating phagocytic cells and those cells belonging to the RE3 system. This localization pattern agrees with the well-documented role of the RE system in the clearance of particulate materials from the circulation. MATERIALS AND METHODS Animals. Specific-pathogen-free C57BL/6N mice were obtained from the Frederick Cancer Research Center's Animal Production Area and the Laboratory Animal Services Division, Smith Kline and French Laboratories. Cell Cultures. The B16-BV8 invasive variant line of the C57BL/6N melanoma (25) was maintained in Eagle's minimal essential medium supplemented with 5% bovine serum, vitamin solution, sodium pyru- vate, nonessential amino acids, and L-glutamine as described previ ously (26). All components of this medium were obtained from Flow Laboratories, Rockville, Md. The medium was endotoxin free as deter mined by the Limulus amebocyte lysate assay (Associates of Cape Cod, Woods Hole, Mass.). All cultures were incubated at 37° in a humidified atmosphere containing 5% CO2 and were free of Myco- plasma, reovirus type 3, pneumonia virus of mice, K virus, Theiler's encephalitis virus, Sendai virus, minute virus of mice, mouse adenovi- rus, mouse hepatitis virus, lymphocytic choriomeningitis virus, ectro- melia virus, and lactate dehydrogenase virus (assayed by MA Bioprod- ucts, Walkersville, Md.) Preparation and Purification of AM. AM were harvested by pulmo nary lavage using a technique slightly modified from that described by Holt (17). Lungs were lavaged from prewarmed endotoxin-free media containing 1x 10~" M dibucaine hydrochloride (Sigma Chemical Co., 3 The abbreviations used are: RE, reticuloendothelial; AM. alveolar macro phages; SUV, small unilamellar, liposomes; MLV, multilameilar, liposomes; [3H]DPPC, [3H]dipalmitoylphosphatidylcholine. 1412 CANCER RESEARCH VOL. 42 on May 24, 2020. © 1982 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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[CANCER RESEARCH 42. 1412-1422, April 1982]0008-5472/82/0042-0000$02.00

Analysis of the Fate of Systemically Administered Liposomes andImplications for Their Use in Drug Delivery1

George Poste,2 Corazón Bucana, Avraham Raz, Peter Bugelski, Richard Kirsh, and Isaiah J. Fidler

Smith Kline and French Laboratories. Philadelphia, Pennsylvania 19101 [G. P., P. B., R. K.¡;Department of Pathology and Laboratory Medicine, University ofPennsylvania, Philadelphia, Pennsylvania 19104 [G. P.]; and Cancer Metastasis and Treatment Laboratory, National Cancer Institute-Frederick Cancer ResearchFacility, Frederick, Maryland 21701 ¡C.B., A. R., I. J. F.]

ABSTRACT

Functional and ultrastructural studies of liposomes injectedi.v. into inbred C57BL/6N mice were performed to determinewhether free liposomes can traverse capillaries. In the liver andspleen, organs with discontinuous (sinusoidal) capillaries, ul-

trastructural and cell fractionation studies revealed that small(300- to 800-Â diameter), sonicated, unilamellar liposomes

were more efficient in penetrating liver sinusoids to interactwith hepatocytes than were large (0.5- to 10-jum) multilamellar

liposomes. Ultrastructural studies of the behavior of liposomesin the continuous capillaries of the lungs revealed that circulating phagocytic cells engulf the liposomes in the capillaries.Transcapillary migration of free liposomes was not observed.We conclude that free liposomes are unable to extravasate toreach the alveoli for subsequent engulfment by alveolar macrophages. Instead, liposomes in the lung capillaries are engulfed by circulating blood phagocytes which subsequentlymigrate to the alveoli to become alveolar macrophages. Experiments on the recruitment of blood monocytes into the lungssubjected to whole- or partial-body X-radiation confirmed thattransfer of i.v.-injected liposomes to the alveolar compartment

was mediated by blood monocytes. The inability of liposomesto escape from continuous capillaries and their rapid uptakeby circulating and fixed phagocytic cells calls into question thefeasibility of using liposomes to "target" drugs to cells in

extravascular tissues.

INTRODUCTION

The ability to selectively "target" cytotoxic drugs to tumor

cells in vivo has been a long-cherished goal in cancer therapy.

In the last few years, liposomes have attracted considerableinterest as potential candidates for accomplishing this task (forreviews, see Refs. 13 and 19). Recent in vitro studies havedemonstrated that liposomes bearing cell-specific antibodiesreact with antigen-bearing target cells with much greater effi

ciency than do liposomes without surface antibodies (14, 15,22). Although encouraging, similar targeting to specific cells invivo has not been successful. Clearly, many obstacles must beovercome to allow liposomes to interact with the desired target

' Research sponsored by the National Cancer Institute, Department of Healthand Human Services, under Contract N01-CO-75380 with Litton Bionetics, Inc.The contents of this publication do not necessarily reflect the views or policies ofthe Department of Health and Human Services, nor does mention of trade names,commercial products, or organizations imply endorsement by the United StatesGovernment.

2 Recipient of USPHS Grants CA18260 and CA30192. To whom requests for

reprints should be addressed, at P. O. Box 7929, Smith Kline and FrenchLaboratories, Philadelphia, Pa. 19101.

Received September 22, 1981; accepted January 4. 1982.

cell. For example, to treat solid tumors or their métastasesthatproliferate in the extravascular compartments of major organs,liposomes containing drugs would be injected into the circulation and thus would have to traverse capillaries to reach thetumor cells (4). If transcapillary passage of intact liposomescannot occur, then the effort of constructing liposomes withsophisticated ligands that "recognize" tumor cells would be

futile because the mechanical barrier imposed by the microcirculation would prevent liposomes from reaching the intendedtarget cell. Information on the transcapillary transport capabilities of liposomes is thus crucial for critical evaluation of thepotential use of liposomes as carriers of cytotoxic drugs incancer therapy.

In this study, we have examined the ability of liposomes ofdiffering size to cross anatomically different classes of capillaries. Our results indicate that limited transcapillary transportof liposomes following i.v. injection occurs in open sinusoidalcapillaries (liver) but does not occur in organs with continuouscapillaries (lung). Moreover, we demonstrated the efficientuptake of liposomes by circulating phagocytic cells and thosecells belonging to the RE3 system. This localization pattern

agrees with the well-documented role of the RE system in the

clearance of particulate materials from the circulation.

MATERIALS AND METHODS

Animals. Specific-pathogen-free C57BL/6N mice were obtainedfrom the Frederick Cancer Research Center's Animal Production Area

and the Laboratory Animal Services Division, Smith Kline and French

Laboratories.Cell Cultures. The B16-BV8 invasive variant line of the C57BL/6N

melanoma (25) was maintained in Eagle's minimal essential medium

supplemented with 5% bovine serum, vitamin solution, sodium pyru-vate, nonessential amino acids, and L-glutamine as described previ

ously (26). All components of this medium were obtained from FlowLaboratories, Rockville, Md. The medium was endotoxin free as determined by the Limulus amebocyte lysate assay (Associates of CapeCod, Woods Hole, Mass.). All cultures were incubated at 37° in a

humidified atmosphere containing 5% CO2 and were free of Myco-plasma, reovirus type 3, pneumonia virus of mice, K virus, Theiler's

encephalitis virus, Sendai virus, minute virus of mice, mouse adenovi-rus, mouse hepatitis virus, lymphocytic choriomeningitis virus, ectro-melia virus, and lactate dehydrogenase virus (assayed by MA Bioprod-

ucts, Walkersville, Md.)Preparation and Purification of AM. AM were harvested by pulmo

nary lavage using a technique slightly modified from that described byHolt (17). Lungs were lavaged from prewarmed endotoxin-free mediacontaining 1 x 10~" M dibucaine hydrochloride (Sigma Chemical Co.,

3 The abbreviations used are: RE, reticuloendothelial; AM. alveolar macro

phages; SUV, small unilamellar, liposomes; MLV, multilameilar, liposomes;[3H]DPPC, [3H]dipalmitoylphosphatidylcholine.

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Liposome-Capillary Interactions

St. Louis, Mo.). Three successive 1,0-ml washes were used, and the

cells which were recovered from each wash were pooled. The lavagedcell suspension was then centrifugea, resuspended in media, andplated into wells of a Microtest II plate with a surface area of 38 sq mm(Falcon Plastics, Oxnard, Calif.). Nonadherent cells (fewer than 10%)were removed by washing with media 1 hr later. Virtually all (>95%) ofthe adherent cells had a typical mononuclear cell morphology, wereable to phagocytose opsonized sheep RBC or latex particles, andstained positively for nonspecific esterase and acid phosphatase.Yields of 1.8 to 2.2 x 106 cells/mouse were obtained routinely.

Preparation and Purification of Liver Cell Populations. The term"nonparenchymal cells" will be used to describe all cells present in

the liver except for parenchymal cells (hepatocytes). The nonparenchymal cell fraction in this paper is the total cell population recoveredfrom the liver after Pronase digestion of parenchymal cells (see below).The nonparenchymal cell fraction includes sinusoidal cells (Kupffercells, endothelial cells), bile duct cells, connective tissue cells, lymphocytes, fat cells, and any other Pronase-resistant cells (e.g., small

numbers of plasma cells, neutrophilic granulocytes, mast cells).C57BL/6N mice given were i.v. injections in the tail vein of radiola-

beled SUV or MLV (phosphatidylcholine:phosphatidylserine, 3:7 molratio; 2 /¿mol/mouse in 0.2 ml phosphate-buffered saline [KCI (0.2 g/

liter), NaCI (8.0 g/liter), KH2PO4 (0.2 g/liter), and Na2HPCv7H2O(2.16 g/liter)]. Livers were removed at the indicated intervals, andpurified fractions of parenchymal and nonparenchymal cells wereprepared (see below). Localization of liposome-associated radioactivity

in each cell fraction was determined as described elsewhere (9) andwas expressed as a percentage of the total radioactivity in the liverbefore fractionation. In experiments with liposomes bearing 2 radiola-bels (membrane-associated [3H]DPPC and encapsulated 5'Cr-EDTA),

the ratio of the 2 radiolabels is normalized by defining the ratio of the2 labels in the original liposome preparation as 1.0, so that ratios arecalculated by simply dividing total radioactivity of one isotope by thetotal radioactivity of the second isotope.

Nonparenchymal cells were isolated by Pronase digestion (21).Typical cell yields of 1.5 x 107 cells/g liver were obtained (liver

weight, 1.46 ±0.22 (S.D.) g; N = 60). This compares favorably with

that reported previously by others (21). Different cell types within theharvested nonparenchymal cells were identified by enzyme cytochemistry and/or cell surface markers. Kupffer cells were identified by theirrapid adherence to glass, positive staining for peroxidase and nonspecific esterase, presence of surface Fc and C3 receptors, and theircapacity to phagocytose 51Cr-labeled sheep RBC or latex beads.

Kupffer cells identified by these criteria were found to represent between 45 and 55% of the total nonparenchymal cell population. Endothelial cells represent the other major cell type in the nonparenchymal fraction (25 to 35%) and were identified by positive esterasestaining and a negative peroxidatic reaction. In certain experiments,purified Kupffer cell and endothelial cell populations were isolated fromheterogeneous nonparenchymal cell fractions by centrifugal elutriation(21 ). Cells were introduced into the rotor at a flow rate of 11.0 ml/minat 4°,and 150-ml fractions were collected at flow rates of 13.5, 20,

22, 25, 31, 37, 40, and 50 ml/min using a rotor speed of 2500 rpm.Kupffer cells were identified by their ability to phagocytose latex beadsor sheep RBC and to stain positively for peroxidase and acid phosphatase. Kupffer cells were recovered primarily in the eluted fractioncollected at a flow rate of 40 ml/min with a smaller number of Kupffercells being recovered at a flow rate of 25 ml/min. Both fractions werecomposed of pure Kupffer cells. Typical yields of 7 to 8 x 106 Kupffer

cells/g liver were obtained with a purity of >90% and a viability of>95%.

Parenchymal cells were isolated by the method of Van Berkel (36).Yields of 5 x 10r cells/g liver with a viability of >95% were obtained

routinely. The parenchymal origin of the recovered cells was confirmedby measurement of 2 hepatocyte-specific enzymes, glutamine synthe-

tase and arginase. The specific activities of both enzymes (milliunits/mg liver protein) measured in isolated parenchymal cells was consis

tently between 80 and 90% of the activities found per mg crude liverhomogenates. The absence of Kupffer cell contamination was confirmed by the failure to detect significant numbers of Fc-positive,esterase-positive phagocytic cells in the preparation.

In all of the procedures described above, no significant differencesin cell yield were found between liposome-treated and untreated control

mice.Lymphokines. Cell-free supernatants were harvested from mitogen-

stimulated rat lymphocytes and from unstimulated control lymphocytesas described previously (26).

Lipids and Preparation of Liposomes. Chromatographically pureegg phosphatidylcholine and beef brain phosphatidylserine were purchased from Avanti Biochemicals, Birmingham, Ala. Sonicated SUVand large MLV were prepared from phosphatidylserine and phosphatidylcholine (3:7 mol ratio) as described elsewhere (9). Liposomepreparations were sized as described in Ref. 9. In typical preparations,more than 90% of the liposomes within any given preparation werewithin the size range of 300 to 800 A and 0.5 to 10 ¡imfor SUV andMLV, respectively. Liposomes containing [3H]DPPC were prepared asdescribed previously (9) by addition of [3H]DPPC (specific activity, 10

Ci/mmol; Applied Science Labs, Inc., State College, Pa.) to the originalphospholipid mixture to give a final specific activity of 0.3 fiCi/mg.Encapsulation of lymphokines (26) and 51Cr-EDTA (9) within liposomes

was achieved as described previously. Liposome preparations alwayswere used within 4 hr. The volume of lymphokine suspension encapsulated within liposomes was determined from measurements of the

aqueous internal space in liposomes as described elsewhere (26).Activation of AM Following i.v. Injection of Free and/or Liposome-

encapsulated Materials. Mice were given i.v. injections of MLV (2.5 or5 /¿molphospholipid) containing lymphokines as described elsewhere(8, 32).

Macrophage-mediated Cytotoxicity Assay. Macrophage-mediated

cytotoxicity was assessed by a radioactive release assay as describedpreviously (26). AM cultures were washed with media, and 5 x 103target cells labeled with 5-['25l]iodo-2'-deoxyuridine were added toeach well in 0.2 ml Eagle's minimal essential medium supplemented

with 5% fetal bovine serum, vitamin solution, sodium pyruvate, nones-sential amino acids, and L-glutamine. Target cells alone were alwaysplated as an additional control. Twenty-four hr after the addition of

target cells, the cultures were washed and refed to remove nonplatedcells. Adherent target cells were lysed with 0.1 ml of 0.2 N NaOH at 24or 72 hr after plating. The lysate was adsorbed on cotton swabs,placed directly into 12- x 75-mm tubes, and monitored for radioactivity

in a gamma counter. The percentage of cytotoxicity in the macrophageassays was computed by the following formula:

% of cytotoxicity

= 100 x

cpm of target cells with normal macrophages—cpm of target cells with activated macrophages

cpm of target cells with normal macrophages

The statistical significance of differences between groups was determined by Student's 2-tailed f test.

X-lrradiation and Bone Marrow Reconstitution. Anesthetized micewere irradiated with 800 rads (source-to-skin distance, 40 cm; doserate, 70 rads/min). X-radiation was given to the whole body, to thewhole body with the thorax and head shielded with 4-mm lead shields,

or to the thorax and head with the remainder of the body shielded. Forreconstitution experiments, bone marrow cells (107) from syngeneicdonors were injected i.v. in 0.2 ml Hank's balanced salt solution at 4

and 24 hr after X-irradiation.

Electron Microscopy. Samples of lung, liver, and spleen wereexcised from mice killed 10 min, 1 hr, 4 hr, and 24 hr after being giveni.v. injections of MLV as described previously (9). Tissues were cut intosmall (>1-cu mm) pieces and fixed in 0.1 M cacodylate buffer (pH 7.3)

containing 3% glutaraldehyde and 2% paraformaldehyde for 1 hr. Thesamples were transferred to a fixative containing 2% glutaraldehyde

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G. Posíeef al.

and 8% fannie acid in cacodylate buffer for 1 hr, rinsed thoroughlywith cacodylate buffer, fixed with 1% buffered osmium tetraoxide for 1hr, rinsed with cacodylate buffer and then with distilled water, andstained en bloc with 1% uranyl acetate for 1 hr. Samples were thendehydrated with a graded series of ethanol. Samples were kept on icethroughout the fixing and dehydration procedures until the last alcoholchanges, at which time the samples were transferred to room temperature. The samples were embedded in Spurr's low viscosity medium

and cut with glass knives on a LKB Ultratome III ultramicrotome.Sections were stained with Reynold's lead citrate for 1 min, dried, and

examined in a Hitachi HU-12A transmission electron microscope at an

accelerating voltage of 75 V.

RESULTS

Fate of i.v. Injected Liposomes in Organs with Discontinuous Capillaries (Liver and Spleen). Problems with fixing andextraction of lipide by solvents during tissue preparation havehindered detection of liposomes in vivo (24, 27). These problems have been overcome by using a tannic acid:glutaraldehyde fixing procedure (20, 38) which enables liposomes to be reliably identified within blood vessels and inextravascular tissues. However, even when well fixed, SUV(300 to 800 A in diameter) are extremely difficult to identify invivo. For this reason, all of the ultrastructural observationspresented here have been derived from experiments usinglarge MLV (0.5 to 10.0 /^m diameter). It is important to notethat glutaraldehyde fixation of cells could lead to the appearance of myelin figures which conceivably may resemble liposomes. There are 2 reasons to rule out such a potential artifact:(a) MLV are much larger than myelin figures; (b) MLV are alsoobserved in freeze fracture of unfixed macrophages that engulfed liposomes.4

Within 10 min after i.v. injection of MLV, electron microscopicobservations revealed liposomes of various sizes within thevascular sinusoids of the liver and the spleen. At this time, nouptake of liposomes into cells was seen (Figs. 1A and 2A). By1 hr, extensive uptake of liposomes by RE cells lining thesinusoids was observed in both the liver (Fig. 18) and thespleen (Fig. 26), but no uptake of MLV by hepatic parenchymalcells and non-RE cells in the spleen was observed. This pattern

remains unchanged even at 4 hr after liposome injection (Figs.1, C and D and 2C). Although 99% of the injected liposomeshave been cleared from the circulation by this time (9), noevidence of liposome uptake by parenchymal cells was detected in either the liver or the spleen.

These ultrastructural observations are supported by studieson the distribution of radiolabeled liposomes in parenchymal(hepatocytes) and Kupffer cell fractions isolated from mouseliver. As shown in Table 1, both SUV and MLV injected i.v.localize initially in the nonparenchymal cell fraction but, withtime, radioactivity is detected in the parenchymal cell fraction.Further separation of the nonparenchymal cell fraction bycentrifugal elutriation (see "Materials and Methods") revealed

that the liposome-derived radioactivity detected in this fraction

localized almost exclusively (>90%) in Kupffer cells (notshown). The fraction of the liposome inoculum recovered inassociation with hepatocytes is higher for SUV than for MLV.Studies using liposomes bearing 2 radiolabels revealed additional differences in the interaction of SUV and MLV with liver

Table 1

Distribution of radiolabeled liposomes in parenchymal and/or Kupffer cells inlivers of mice given i. v. injections of SUV and ML V

C57BL/6N mice were given i.v. injections in the tail vein of radiolabeled SUVor MLV (phosphatidylcholine:phosphatidylserine, 7:3 mol ratio; 2 /¿mol/mouse in0.2 ml phosphate-buffered saline). Livers were removed at the indicated intervals,and purified fractions of parenchymal (hepatocytes) and Kupffer cells (RE) wereprepared. The cell-associated radioactivity ([3H]DPPC and s'Cr-EDTA) was mea

sured in each fraction and was expressed as a percentage of the total radioactivityin the liver before fractionation. In experiments with liposomes bearing 2 radio-labels, the ratio of the 2 radiolabels has been normalized by defining the ratio ofthe 2 labels in the original liposome preparation as 1.0 so that ratios in the liverfraction were calculated by dividing total radioactivity of one isotope by the totalradioactivity of the second isotope. The results represent mean values frommeasurements on 20 mice at each time interval for both types of liposomes.

% of liver-associated radioactiv

ity in cell populationst[3H]DPPC)

Ratio of membrane-associated([3H]DPPC) and encapsulated

(51Cr-EDTA) radiolabels

SUV MLV SUV MLV

Paren- Paren- Paren- Paren-Time chymal RE chymal RE chymal RE chymal RE

10min1hr4hr8hrNDa14.530.731.381.365.855.945.4ND5.611.414.182.476.369.361.6ND1.322.743.051.271.411.481.93ND5.917.648.271.241.241.831.64

4 C. Bucana and L. Hoyer, manuscript in preparation.

ND, not detected.

cells. The ratio of membrane-associated to encapsulated radi

olabels detected in the nonparenchymal cell fraction was similar to that in the original inoculum (1.0) for both SUV and MLV,indicating that the liposomes were structurally intact. In contrast, the ratio in the parenchymal fraction was increased,particularly for MLV. This indicates that loss of liposomal contents has occurred. In the case of MLV, the marked increasein the ratio and the failure to detect liposomes in hepatocytesby electron microscopy suggest that transfer of radiolabeledphospholipid to these cells may occur by an exchange process.These results could not be attributed to an artifact in the cellisolation procedure since the samples for the MLV and SUVstudies were prepared in an identical manner and both the SUVand MLV were prepared from identical phospholipids. Thus,intact liposomes do not penetrate the sinusoidal lining to interact with hepatocytes on any significant scale.

Fate of i.v.-injected Liposomes in Organs with Continuous

Capillaries (Lung). In contrast to the discontinuous capillariesin liver and spleen, the capillaries in other major organs suchas the lung possess a continuous endothelium and basal lamina(42). To study whether liposomes can successfully traverse thewall of continuous capillaries, we examined the behavior of i.v.-injected liposomes in the lung capillary bed. The lung waschosen not only because of the structure of its capillaries butalso because of our interest in using liposomes as carriers fordelivering immunomodulators to the lung to stimulate antitumoractivities in AM (8-10). Previous studies from our laboratories

have shown that, following i.v. injection of MLV, liposomes arepresent in AM recovered from the alveoli by lavage (8, 10, 32).Whether the uptake of MLV by AM occurs in the alveolar spacefollowing extravasation of liposomes or whether MLV wereengulfed by blood monocytes which then migrated to the alveolito become AM was investigated.

The arrest of MLV in the pulmonary capillary bed after i.v.injection (Fig. 3, A and 6) is followed by their uptake into bloodmonocytes (Fig. 3, C and D) or into polymorphonuclear cells(Fig. 4). By 4 hr after injection, mononuclear cells containingliposomes were also detected in the alveolar spaces (Fig. 3£).

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AM containing engulfed MLV can be reliably distinguished fromtype II pneumocytes. The lipid bilayers of MLV appear asdistinct circular or elliptical membranes, whereas inclusions oflung surfactant in type II pneumocytes contain stacks of lipidlamella rather than concentric whorls (Fig. 3F). The presenceof liposomes free in the alveolar tissues, or in any other location, suggesting transcapillary transfer of free liposomes wasnever observed. On the other hand, the high frequency ofliposomes within blood monocytes and/or polymorphonuclearcells suggests that liposomes reach the extravascular compartment by passive transfer inside these cells during theirmigration from the bloodstream into the alveoli.

Additional evidence that liposomes are unable to cross lungcapillaries and that they reach the alveoli only when transportedinside migrating cells has been obtained from our experimentson the recruitment of AM to the lung. In the normal lung, AMare derived from blood monocytes of bone marrow origin (2, 3,12, 33, 37, 38). Whole-body X-irradiation suppresses circulat

ing monocytes and, by damaging precursors in the bone marrow, X-irradiation also prevents the recruitment of monocytesto the lung (2, 37). Reconstitution of X-irradiated animals with

the bone marrow cells results in reappearance of AM in lunglavage fluids within 7 to 10 days (37). In contrast, localized X-

irradiation of the chest has been shown to damage preexistingAM, but this can be rapidly compensated by recruitment ofblood monocytes into the alveoli (37). We have exploited theseconditions to determine the origin of tumoricidal AM recoveredfrom mice given i.v. injections of liposomes containing encapsulated lymphokines. As reported in our previous work (8, 32),AM harvested from normal rodents (mice or rats) do not exhibittumoricidal activity. The i.v. injection of liposome-encapsulated

lymphokines results in the activation of AM to become cytotoxicagainst neoplastic cells (Table 2). Whole-body X-irradiation ofmice 24 hr before the i.v. injection of liposomes containing

lymphokines abolished the in vivo activation of AM (Table 2).This experiment was complicated, however, by the fact thatfollowing whole-body X-irradiation very few viable cells can berecovered from the lung. Lavage fluids from X-irradiated mice

(800 rads) primarily contained dead cells and debris. Mice inwhich the thorax was shielded during irradiation with 800 radsof X-radiation showed a similar loss of responsiveness by AMto in situ activation by i.v.-injected liposomes (Table 2). The

yield of lavaged AM from these mice was similar to that recovered from untreated controls (data not shown). We do notattribute the unresponsiveness of these AM to i.v.-injected MLV

containing lymphokines to their inability to respond to theactivation stimulus. Data shown in Table 2 demonstrate thatAM recovered from X-irradiated mice can be activated by

incubation in vitro with liposomes containing lymphokines.Shielding of the thorax during irradiation protects the AM butdamages circulating monocytes and macrophage precursorsin the bone marrow (37). The failure of the protected AM torespond to liposome-encapsulated lymphokines in vivo sug

gests that liposomes are simply not reaching these cells invivo. In contrast, mice subjected to local X-irradiation of thechest before the i.v. injection of liposome-encapsulated lym

phokines yielded AM with a significant tumoricidal activity(Table 2). X-irradiation of the thorax destroyed macrophagesin the alveoli but had little effect on circulating monocytes ortheir recruitment to the lung. These data strengthen the hypothesis that AM are derived from blood monocytes that interact with liposomes within the microcirculation and then migrateinto the alveoli. The recovery of the response of AM to liposome-encapsulated lymphokines in mice given whole-body X-

radiation (800 rads) following reconstitution with bone marrowcells also supports this interpretation. AM from mice treatedwith whole-body X-irradiation are unresponsive to activation byliposomes containing lymphokines injected 1 day after recon-

Table 2Effects of partial or whole-body X-irradiation on the tumoricidal activity of AM harvested from mice given

i.v. injections of liposomes containing encapsulated lymphokines

Treatment of macrophagedonorsX-irradiation

(800rads)NoneNoneWhole-bodyWhole-body

thoraxshieldedThorax

X-irradiatedWhole-body

bone marrow cellreconstituted'Whole-body,

bone marrow cellreconstituted'Time

of i.v. injectionofliposomesNone24

hrbeforeharvestof AM

24 hr afterX-irradiation

24 hr afterX-¡rradiation24

hr afterX-irradiation

24 hr afterreconstitution7

days afterreconstitution%

of destruction of B1 6 melanoma cells byAMaIn

vivo activationwith*MLV639o"8"32o*30SUV0310*5eNot

doneNot

doneNot

doneIn

vitro incubationwith0MLV4453Not

done4749Not

done42SUV037Not

done39Not

doneNot

doneNot

done

a AM (1x105) were plated into 38-sq mm wells. The AM were thoroughly washed before the additionof 5-{'25l]iodo-2'-deoxyuridine-labeled 616 melanoma cells. Percentage of cytotoxicity was calculated by

comparison to untreated AM. Means of triplicate cultures terminated after 72 hr of cocultivation. This is arepresentative of several experiments.

6 Mice were given i.v. injections of 5 ^mol phospholipids (MLV or SUV).0 AM harvested from the various treatment groups were incubated in vitro with SUV, with MLV containing

lymphokines, or with media alone (controls) for 24 hr. The AM cultures were then thoroughly washed, andtheir tumoricidal activity was monitored against the radiolabeled B16 target cells.

d No viable cells were recovered.8 Decrease in tumoricidal properties differed significantly from the appropriate control groups (p <

0.001).Bone marrow cells (107/mouse) were injected i.v. at the indicated time.

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G. Poste et al.

stitution with bone marrow cells. However, significant tumori-cidal response of AM is observed following the i.v. injectioninto mice of MLV containing lymphokines reconstituted withbone marrow cells 7 days previously (Table 2).

DISCUSSION

To date, morphological information on the intravascular behavior and fate of i.v.-injected liposomes has been limited to

ultrastructural observations showing that components fromMLV could be taken up by both Kupffer cells and hepatocytes(7, 29, 30, 44). Substantial technical difficulties in preservingliposomal structure for electron microscopy have hamperedinvestigation of the fate of liposomes after their arrest in thecapillary beds of other major organs. The tannic acid:glutaraldehyde procedure used here provides both excellentfixing of lipids and resistance to solvent extraction (20). Thistechnique is therefore far superior to the usual method ofstabilizing liposome structure by fixing with osmium. Tannicacid:glutaraldehyde also increases the electron density of thesurface membrane of capillary endothelial cells (38), thus facilitating the study of liposome-endothelial interactions. Finally,

the excellent lipid fixing achieved with this method makes itpossible to distinguish liposomes incorporated into AM fromthe lamellar inclusions of surfactant present in type II pneu-

mocytes (Fig. 3F).The ultrastructural experiments described here have failed

to detect any evidence of transcapillary passage of MLV to theextravascular compartments in the liver, spleen, or lung. Mechanically, opportunities for transcapillary transfer of circulating liposomes would be expected to be greatest in the sinusoidsof the liver and spleen. In these organs, the endothelium isdiscontinuous and is interrupted by large gaps. Moreover, thesubendothelial basement membrane in these capillaries is absent (42). The mean diameter of endothelial openings in thesinusoidal wall is 0.1 ¡im,and particles of larger diameter fail togain access to hepatocytes (16, 43). The elastic deformabilityof liposomes (11) may permit liposomes marginally larger than0.1 jum to penetrate endothelial gaps to interact subsequentlywith hepatocytes. However, it is extremely unlikely that intact,large MLV (0.5 to 10 /¿m)could penetrate through the endothelial barrier.

Our analysis of liposome localization in nonparenchymal,sinusoidal, and parenchymal liver cells reveals that liposomesdo indeed gain access to hepatocytes. The kinetics of liposomeassociation with hepatocytes is slower than with the nonparenchymal (RE) cell fraction. SUV are more efficient than areMLV in interacting with hepatocytes. This may simply reflectthe ability of SUV to penetrate endothelial gaps. Furthermore,our experiments with the double-labeled liposomes indicate

that, whereas SUV recovered in association with hepatocytesare apparently intact, MLV retained in the liver show preferential transfer of liposomal lipid to hepatocytes without theconcomitant transfer of liposomal contents. Similar results havebeen reported recently by Preise ef al. (11 ).

In contrast to the transfer, albeit limited, of liposomal components across hepatic sinusoids, our results indicate that lungcapillaries are completely impermeable to either SUV or MLV.This finding is perhaps not surprising when interpreted in termsof our current knowledge of capillary structure. Lung capillariespossess an uninterrupted lining of endothelial cells with tight

junctions between adjacent cells which, in turn, overlie a continuous basal lamina. Studies using tracer molecules of definedsize indicate that endothelial junctional permeability is restricted to particles smaller than 20 A in diameter (31). Thesystem of endothelial vesicles, which are believed to representthe so-called "large-pore" permeability channel in continuous

capillaries (4), do not exceed 800 A in diameter and would notaccommodate the MLV (0.5 to 10 /um) observed in AM in thisstudy. Moreover, recent studies indicate that endothelial vesicles may not be a dynamic system in which vesicles arecontinually formed but may represent a permanent structuralchannel crossing the endothelial cell (4). If this is the case,then the limited diameter (20 A) of the "neck" of vesicles

opening to the luminal surface would be far too small toaccommodate even SUV.

Even if arrest of liposomes in the pulmonary microcirculationwere somehow to lead to dissolution of endothelial cell junctions and to permit the liposomes to penetrate between adjacent cells, liposomes must still cross the subendothelial basallamina and the adventitial connective tissue and penetratebetween alveolar epithelial cells to eventually reach the alveolarspace. For this reason, it is likely that continuous capillaries ofthe kind found in the lung and in the central nervous system,muscle, connective tissue, exocrine pancreas, and gonads (42)present an impenetrable mechanical barrier to the movementof circulating liposomes into the extravascular tissues. Theimpenetrability of brain capillaries to liposomes has been demonstrated by the failure of potent centrally acting neurotropicagents encapsulated in liposomes to alter brain function afteri.v. injection, even under conditions in which permeability ofthe blood-brain barrier to serum albumin is markedly enhanced

(35). Reports that SUV administered i.v. increase brain glucoseand the release of catecholamine (24) do not establish thatliposomes can cross brain capillaries. What is more likely isthat these actions are attributable to the well-documentedability of exogenous phospholipids to alter cerebral metabolismand catecholamine turnover (5, 6).

The studies presented here on the recruitment and transcapillary migration of blood monocytes into the alveolar spaceindicate that liposomes can be transferred passively acrosscapillaries by being carried inside migratory cells. These observations are also pertinent to the mechanisms by whichliposome-encapsulated macrophage activators stimulate mac-rophage-mediated tumoricidal reactions in the lung (8-10, 32).

Our results suggest that the population of tumoricidal AMrecovered from alveoli after i.v. injection of liposomes containing macrophage activators are derived from circulating monocytes that engulf liposomes within pulmonary capillaries andsubsequently migrate to the extravascular alveolar tissue.

The interaction of liposomes with a third class of capillaries,the fenestrated capillaries, has yet to be investigated in detail.However, preliminary observations from our laboratory on thetransfer of liposomes across fenestrated capillaries in the intactisolated perfused cat salivary gland have failed to detect significant transfer of either SUV or MLV5.

Ever since the first reports describing the use of liposomesas drug carriers were published, investigators have discussedthe possibility of using liposomes to target drugs to specific cell

5 G. Poste, P. Bugelski, R. Kirsh, P. Sears, and G. Waterson, manuscript in

preparation.

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Liposome-Capillary Interactions

types in vivo. Unfortunately, our findings that circulating liposomes localize in phagocytic mononuclear cells and that liposomes have limited ability to traverse capillaries pose majorobstacles to the targeting of liposomes to organ parenchymalcells situated outside the circulation. Unless methods can bedevised to render capillaries selectively permeable to liposomes without impairing their permeability to blood cells orother materials, the data presented here suggest that theprobability of liposomes reaching extravascular target cells willbe low, even in tumors vascularized by capillaries with structural defects (23).

Although the present findings raise serious doubts about thefeasibility of targeting liposomes to target cells in the extravascular compartment, we consider that targeting of liposome-

encapsulated materials within the confines of the intravascularcompartment still offers exciting therapeutic opportunities. Forexample, it should be possible to direct ligand-bearing lipo

somes to specific blood cells (e.g., lymphoid cell subsets) andperhaps also to target liposomes to specific regions of thevascular system using ligands directed against regional and/or organ-specific determinants on the vascular lumen (28). The

ability to achieve high concentrations of drugs in selectedorgans by such an approach could be of substantial therapeuticvalue. This approach might also be combined profitably withthe use of the recently described novel classes of liposomeswhich break down and release their contents in response tochanges in the physicochemical properties of the local tissueenvironment, such as temperature and pH (41, 45).

In conclusion, the mechanical barrier of the capillary wall isa major hindrance to the targeting of liposomes to many cells.In contrast, the localization of liposomes in circulating and fixedphagocytic cells offers a viable mechanism for targeting lipo-some-associated materials to RE cells and monocytes. This

strategy has already been used successfully to deliver materials which stimulate macrophage-mediated defense reactions

against parasites (1 ) and tumors (9, 10).

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1. Alving, C. R., and Steck, E. A. The use of liposome-encapsulated drugs inLeishmaniasis. TIBS, 4: 175-177, 1979.

2. Blusse Van Oud, A., and Van Furth, R. Origin, kinetics and characteristicsof pulmonary macrophages in the normal steady state. J. Exp. Med., Õ49.1504-1518, 1979.

3. Brunstetter, M. A., Handie, J. A., and Schiff, R. The origin of pulmonaryalveolar macrophages: studies of stem cells using the ES-2 marker of mice.Arch. Intern. Med., 127: 1064-1068, 1971.

4. Bundgaard, H. Transport pathways in capillaries—in search of pores. In: I.S. Edelman (ed.), Annual Review of Physiology, pp. 325-336. Palo Alto.Calif.: Annual Reviews, Inc., 1980.

5. Casament, F., Mantovani, P., Amaducci, L., and Pepeu. G. Effect of phos-phatidylserine on acetylcholine output from the cerebral cortex of the rat. J.Neurochem., 32: 529-533, 1979.

6. Cohen, E. L., and Wertman, R. J. Brain acetylcholine: control by dietarycholine. Science (Wash. D. C.). T9Õ.561-562, 1976.

7. De Barsy, T., Devos, P., and Van Hoof. F. A morphologic and biochemicalstudy of the fate of antibody-bearing liposomes. Lab. Invest., 34: 273-282,1976.

8. Fidler, I. J., Hart, I. R.. Raz. A., Fogler, W. E., Kirsh, R., and Poste, G.Activation of tumoricidal properties in macrophages by liposome-encapsulated lymphokines: in vivo studies. In: B. H. Tom and H. Six (eds.). Liposomesand Immunobiology, pp. 109-118. Amsterdam: Elsevier/North Holland Pub

lishing Co., 1980.9. Fidler, I. J., Raz, A., Fogler, W. E., Kirsh, R., Bugelski, P., and Poste, G.

Design of liposomes to improve delivery of macrophage-augmenting agentsto alveolar macrophages. Cancer Res., 40 4460-4466. 1980.

10. Fidler, I. J., Soné,S., Fogler, W. E.. and Barnes, Z. Eradication of sponta

neous métastasesand activation of alveolar macrophages by intravenousinjection of liposomes containing muramyl dipeptide. Proc. Nati. Acad. Sci.U. S. A., 78: 1680-1684, 1981.

11. Preise, J., Muller, W. H., Brolsch, C. H., and Schmidt, F. W. In vivodistribution of liposomes between parenchymal and nonparenchymal cellsin rat liver. Biomedicine (Paris), 32. 118-123, 1980.

12. Godleski, J. J., and Brain, J. D. The origin of alveolar macrophages in mouseradiation chimeras. J. Exp. Med.. 136: 630-347, 1972.

13. Gregoriadis, G., and Allison, A. C. (eds.). Liposomes in Biology and Medicine, New York: Wiley Interscience, 1980.

14. Gregoriadis, G., Neerunjun, D. E., and Hunt, R. Fate of liposome-associatedagents injected into normal and tumour-bearing rodents: attempts to improvelocalization in tumour lines. Life Sci., 21: 357-370, 1977.

15. Heath, T. D., Macher, B. A., and Papahadjopoulos, D. Covalent attachmentof immunoglobulins to liposomes via glycosphingolipids. Biochim. BiophysActa, 640. 66-81, 1981.

16. Hoekstra, D., Tomasini, R., and Scherphof, G. Interaction of phospholipidvesicles with rat hepatocytes in primary monolayer culture. Biochim. Biophys. Acta, 542. 456-469, 1978.

17. Holt, P. G. Alveolar macrophages. I. A simple technique for the preparationof high number of viable alveolar macrophages for small laboratory animals.J. Immunol. Methods. 27: 189-198, 1979.

18. Johnson, K. J., Ward. P. A., Striker, G., and Kunkel. R. A study of the originof pulmonary macrophages using the Chediak-Higashi marker. Am. J. Pa-thol., 101: 365-368, 1980.

19. Juliano, R. (ed.). Drug Delivery Systems. Oxford, England: Oxford UniversityPress, 1980.

20. Kalina, M., and Pease, D. C. The preservation of ultrastructure in saturatedphosphatidylcholines by tannic acid in model systems and type H pneumo-cytes. J. Cell Biol., 74: 726-741, 1977.

21. Knock, D. L., and Sleyster, E. Preparation and characterization of Kupffercells from rat and mouse liver. In: E. Wisse and D. L. Knook (eds.), KupfferCells and Other Liver Sinusoidal Cells, pp. 273-288. Amsterdam: Elsevier/North-Holland Publishing Co., 1977.

22. Leserman, L. D., Weinstein, J. N., Blumenthal, R., and Terry, W. D. Receptor-mediated endocytosis of antibody-opsonized liposomes by tumor cells. J.Cell Biol., 77. 4089-4093, 1980.

23. Peterson. H.-l. (ed.). Tumor Blood Circulation: Angiogenesis. Vascular Morphology and Blood Flow of Experimental and Human Tumors. Boca Raton,Fla.: CRC Press, Inc.. 1979.

24. Poste, G. The interaction of lipid vesicles (liposomes) with cultured cells andtheir use as carriers for drugs and macromolecules: In: G. Gregoriadis andA. C. Allison (eds.), Liposomes in Biology and Medicine, pp. 101 -151. New

York: Wiley Interscience, 1980.25. Poste, G., Doll, J., Hart, I. R., and Fidler, I. J. In vitro selection of murine

B16 melanoma variants with enhanced tissue-invasive properties. CancerRes., 40: 1636-1644, 1980.

26. Poste, G., Kirsh, R., Fogler, W. E., and Fidler, I. J. Activation of tumoricidalproperties in mouse macrophages by lymphokines encapsulated in liposomes. Cancer Res., 39. 881-892, 1979.

27. Poste, G., Porter, C.W., and Papahadjopoulos, D. Identification of a potentialartifact in the use of electron microscope autoradiography to localize saturated phospholipids in cells. Biochim. Biophys. Acta, 570. 256-263, 1978.

28. Pressman, D. The development and use of radiolabeled antitumor antibodies.Cancer Res.. 40. 2960-2964. 1980.

29. Rahman, Y. E., and Wright, B. J. Liposomes containing chelating agents. J.Cell Biol., 65. 112-122, 1975.

30. Segal, A. W., Wills, E. J., Richmond, J. E., Slavin. G., Black. C. D. V.. andGregoriadis, G. Morphological observations on the cellular and subcellulardestination of intravenously administered liposomes. Br. J. Exp. Pathol.. 55.320-327, 1974.

31. Simionescu, N., Simionescu, M., and Palade, G. E. Permeability of musclecapillaries to small heme-peptides—evidence for the existence of patenttransendothelial channels. J. Cell Biol., 64: 586-607, 1975.

32. Soné,S., Poste, G., and Fidler, l. J. Rat alveolar macrophages are susceptible to activation by tree and liposome-encapsulated lymphokines. J. Immunol., 124: 2197-2202, 1980.

33. Thomas, E. D., Ramberg, R. E.. Sale, G. E., Sparkes, R. S., and Golde, D.W. Direct evidence for a bone marrow origin of the alveolar macrophage inman. Science (Wash. D.C.), Õ92. 1016-1018, 1976.

34. Tuffano, G., and Bruni, A. Pharmacological properties of phospholipid liposomes. Pharmacol. Res. Commun., 12: 829-845, 1980.

35. Tokes, Z. A., Kulcsar St. Peteri, A., and Todd, J. A. Availability of liposomecontent to the nervous system. Liposomes and the blood-brain barrier. BrainRes., 188. 282-286, 1980.

36. Van Berkel, T. J. C. Identity and activities of lysosomal enzymes in parenchymal and nonparenchymal cells from rat liver. Eur. J. Biochem., 58: 145-

152. 1975.37. Velo, G. P., and Spector, W. G. The origin and turnover of alveolar macro

phages in experimental pneumonia. J. Pathol, 709. 7-19, 1973.38. Wagner. R. C. The effect of tannic acid on electron images of capillary

endothelial cell membranes. J. Ultrastruct. Res., 57: 132-139, 1976.39. Weiden, P. L.. Storb. R.. and Tsoi, M-S. Marrow origin of canine alveolar

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macrophages. J. Reticuloendothel. Soc., / 7. 342-345, 1975. 43. Wisse, E. An electron microscopie study of the fenestrated endothelium40. Weinstein, J. W., Blumenthal, R., Sharrow, S. O., and Henkart, P. A. lining of rat liver sinusoids. J. Ultrastruct. Res., 31: 125-142, 1970.

Antibody-mediated targeting of liposomes: binding to lymphocytes does not 44. Wisse, E., Gregoriadis, G., and Daems, W. T. Electron microscopic locali-ensure incorporation of vesicle contents into cells. Biochim. Biophys. Acta, zation of intravenously injected liposome-encapsulated horseradish peroxi-509. 272-279, 1979. dase in rat liver cells. In: S. M. Reichard, M. Escobar, and H. Friedman

41. Weinstein, J. W., Magin, R. L.. Yatvin, M. B., and Zaharko. M. B. Liposomes (eds.). The Reticuloendothelial System in Health and Disease, pp. 307-326.and local hyperthemia: selective delivery of methotrexate to heated tumors. New York: Plenum Publishing Corp., 1976.Science (Wash. D. C.), 204. 188-191, 1979. 45. Yatvin, M. B., Krentz, W., Horwitz, B. A., and Shinitzky. M. Ph-sensitive

42. Weiss, L., and Greep, R. O. (eds.). Histology, pp. 398-415. New York: liposomes: possible clinical implications. Science (Wash. D. C.), 2/0. 1253-McGraw-Hill Book Co., 1977. 1255, 1980.

Fig. 1. Electron micrographs of mouse liver after the i.v. injection of MLV. In A, liposomes (arrows) are visible in liver sinuses 10 min after being injected i.v. In B,small MLV are seen phagocytosed by Kupffer cells 1 hr after being injected i.v. C, MLV in liver sinus adjacent to RBC. Liver hepatocytes (H) are indicated at theperiphery of the sinus. D, MLV phagocytosed by Kupffer cells. Portions of liver hepatocytes (H) are also shown.

Fig. 2. Electron micrographs of mouse spleens after i.v. injection of MLV. A, sample taken 10 min after i.v. injection shows numerous liposomes (arrows) in theblood sinus. B, sample taken 1 hr after i.v. injection. C, sample taken 4 hr after i.v. injection. Note the uptake of liposomes by splenic phagocytes.

Fig. 3. Electron micrographs of mouse lung after i.v. injection of MLV. In A, liposomes (arrows) of various sizes are visible in the lung capillary 10 min after i.v.injection. B, large liposomes (arrows) visible inside the capillary 1 hr after i.v. injection. C. large MLV in the capillary 60 min after i.v. injection. P, polymorphonuclearcell; M, monocyte. D, monocyte with internalized MLV 60 min after i.v. injection. Note the presence of RBC in the lumen. £,AM with internalized liposome. Theliposome is adjacent to the nucleus (A/) of the macrophage. The capillary lumen is indicated by arrows. F, pneumocyte type II from untreated mouse. Note the well-preserved ultrastructure of the surfactant (arrows) accentuated by the use of the fannie acid:glutaraldehyde fixing technique.

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,•:;!

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G. Poste et al.

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Liposome-Capillary Interactions

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G. Posfe et al.

Fig. 4. Uptake of liposomes by circulatingpolymorphonuclear cells. Leukocytes were isolated from the blood of mice given i.v. injections ofMLV 60 min earlier. Arrows, liposomes.

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1982;42:1412-1422. Cancer Res   George Poste, Corazon Bucana, Avraham Raz, et al.   and Implications for Their Use in Drug DeliveryAnalysis of the Fate of Systemically Administered Liposomes

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