[CANCER RESEARCH54. 3352—3356,July 1, 19941

Advances in Brief

Microvascular Permeability and Interstitial Penetration of Sterically Stabilized(Stealth) Liposomes in a Human Tumor Xenograft'

Fan Yuan, Michael Leunig, Shi Kun Huang, David A. Berk, Demetrios Papahadjopoulos, and Rakesh K. Jam

Department of Radiation Oncology, Massachusetts General Hospital and Harvard Medical Schoo4 Boston, Massachusetts 02114 [F. Y., M. L., D. A. B., R. K. J.j, and CancerResearch Institute and Department of Pharmacology, University of California, San Francisco, California 94143 [5. K H., D. P.J

reveal heterogeneous distribution of liposomes in tumor interstitium,and suggest possible pathways of transendothelial transport.

Materials and Methods

Animal and Tumor Model. The in vivo model of both normal and tumormicrocirculationis describedby Leuniget aL (9). Briefly,the titaniumchambees were implanted on the dorsal skin of SCID mice and then a 2-pi densesuspension (—2 x 10' cells) of human colon adenocarcinoma LS174T cellswas inoculated onto the striated muscle layer of the s.c. tissue in chambers. Theexperiments were performed between days 15 and 32 after tumor cell implan

tation.Liposomes and Tracer Molecules. The liposomes were prepared using the

same procedure as used by Huang et a!. (5) and fluorescently labeled byRho-phosphatidylethanolamine (Molecular Probes, Eugene, OR) with excitation/emission wavelengths of 546/590 ant. The molar ratio of egg phosphatidylcholine:cholesterol:polyethylene glycol conjugated with distearoylphosphatidylethanolamine:Rho-phosphatidylethanolamine was 10:5:0.8:0.1, and

the average diameter of the liposomes was between 86 and 90 nm as determined by dynamic light scattering (5). The fluorescently labeled liposomeswere then diluted with physiological saline at a concentration of 10 mMlipid.As a control, extravasation of Rho-labeled BSA (A847; Mr 67,000; MolecularProbes)was also studied. Rho-BSA was dissolved in phosphate-buffered saline(lx; Sigma Chemical Co., St. Louis, MO) at a concentration of 8 mg/ml. Thefree fluorescent dye in the solution of Rhodamine-labeled SL and BSA wasremoved by passing the solution through size exclusion columns (2, 8).

Experimental Procedure. The experimental procedure is similar to ourprevious studies (8, 9). In brief, male mice weighing 25-30 g were anesthetizedby s.c. injection of Ketamine and Xylazine (7.5 mg ketamine hydrochlorideand 2.5 mg xylazine per 100 g body weight) and put in a polycarbonate tubewhich provides rigid support for holding the chamber on a polycarbonatestage placed under an intravital fluorescence microscope (Axioplan, Zeiss,Oberkochen, Germany) equipped with the fluorescence filter set for Rho

(Omega Optical, Inc., Brattleboro, VT), an intensified CCD video camera(C2400—88;Hamamatsu Photonics K.K., Hamamatsu, Japan), a photomultiplier (9203B; EM!, Rockaway, NJ), and a Nikon camera (HFX-DX,Melville, NY). A heating pad was used to maintain the skin temperature(32°C) of mice (9).

Animals were divided into two groups. In the first group, the extravasationof SL (n = 23) and BSA (n = 6) in tumors was measured. In each experiment,the background intensity under epifluorescence illumination (Mercury Lamp,100 W) was first measured by the photomultiplier. Then a bolus injection ofeither SL suspension or BSA solution (0.1 ml per 25 g body weight) was givenduring a 20-s period through the tail vein. The fluorescence from the tissue wasmeasured intermittently by the photomultiplier, with the time interval for eachobservation of less than 10 s to avoid fluorescent molecule-mediated photochemical toxicity to endothelial cells (8). At the end of the experiments, thefluorescence images of vasculature were recorded on a video tape (videocassette recorder, AG-6500; Panasonic, Secaucus, NJ) for offline analysis of

vessel diameter and length (8). The total time interval for permeability mea

surements was 10 and 40 min for BSA and SL, respectively, whereas liposomeaccumulation in tumors was monitored for up to 1 week.

In the second group, the extravasation of liposomes in normal s.c. tissue(n 12) was monitored intermittentlyfor up to 2 weeks after SL injection. In4 of 12 chambers, the s.c. tissue was superfused (topical application) withhistamine solution (10@ M)after liposome injection. The procedure of super

Abstract

Microvascular permeability and interstitial penetration of stesicaHystabilized liposomes In both normal s.c. tissue and human colon adenocarcinoma LS174T xenograft were quantified by using the dorsal skin

fold chamber implanted in severe combined Immunodeficient mice andintravital fluorescence microscopy. Significant extravascular accumula

tion was the dominant feature ofliposome distribution Intumors, whereasonly minimal Intramural accumulation in postcaplllary and collectingvenules was observed in normal s.c. tissue. The extravasated liposomes intumors distributed heterogeneously and formed perivascular clusters thatdid not move significantly and could be observed for up to1 week. The effective permeability of tumor vessels to liposomes(2.0±1.6x i0@ cm/s;n = 23)wassixtimessmallerthanthat to bovineserum albumin (1.2 ±0.5 x i0@ cm/s; n 6). These results provide newinsights into the mechanisms of transendothelial pathways of liposomesand Improvements In liposome-medlated drug delivery.

Introduction

@ have shown improved localization of drugs in tumors (1).

Recent therapeutic studies with SL-encapsulated doxorubicin demonstrated a substantially increased efficacy of this drug against severaltumors implanted in mice (2—4).These tumors are generally resistantto doxorubicin as a free drug or when encapsulated in conventionalliposomes. The improvement of the therapeutic efficacy has beenattributed to the prolonged blood half-life and enhanced tumor uptakeof drugs. However, mechanisms of liposome extravasation and theirinterstitial movement are still unknown. These mechanisms havedirect implications on strategies of liposome-mediated tumor therapy,since liposome extravasation and their interstitial movement affectmicroscopic distribution of liposomes, which in turn determines possible targets of liposome-encapsulated drugs in tumors. Previousstudies have shown that SL can extravasate in tumors (1, 5—7),butpathways of transvascular transport of liposomes are not understood.In addition, no information on liposome penetration in tumor interstitium has been provided in literature. Thus, the current study was

designed to investigate the mechanisms of liposome extravasation andtheir interstitial movement. The unique features of our approach werenoninvasive measurement of SL extravasation (8) and dynamic observation of SL accumulation in both normal and tumor tissue indorsal skinfold chambers implanted in SCID mice (9). The results ofthis study provide quantitative information on the extravasation of SL,

Received 3/7/94; accepted 5/19/94.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by R35-CA-56591. Presented at the 86th Annual Meeting of AmericanInstitute of Chemical Engineers, 1993; 85th Annual Meeting of the American Associationfor Cancer Research, 1994; Annual Meeting of Microcirculation Society, 1994; and 42ndAnnualMeetingof RadiationResearchSociety,1994.M.L wasa recipientofthe FenderLynen fellowship from the Humboldt Foundation (1991—1993).D. A. B. is a recipient ofa National Research Service Award (CA59255).

2The abbreviations used are: SL, stencally stabilized (Stealth) liposome; Rho, rhodamine; BSA, bovine serum albumin; SCID, severe combined immunodeficient.

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LIPOSOME DELIVERY IN TUMORS

fusion was as follows. The glass cover slip of the chamber was first removed,and 50 ILlphysiological saline was added on the top of s.c. tissue. Then thechamber was put on the stage of the microscope, and 50 p.1histamine solutionwas topically applied to the chamber. The fluorescence image and intensity ofnormals.c. tissuewere recordedand measured,respectively,bothbeforeandafter histamine superfusion. At the end of the experiment, the chamber wasremoved from the stage of the microscope and sealed again by a new sterilizedcover slip.

Estimation of Vascular Permeability. To minimize intratumor heteroge

neity of vascular structure, we measured the effective microvascular permeability (P) averaged over a circular area of 1.3 mm in diameter. Thus, P in thisstudy depends not only on microvascular permeability but also on the ratio ofthe number of leaky versus nonleaky vessels in that area. If the time intervalfor permeability measurement (10 and 40 min for BSA and SL, respectively)is much shorter than the time constant of plasma clearance (K), the equation

to estimate P is:

V/id! 1P=(l-HT)@y-@+@

derived by Yuan et aL (8), where I is the average fluorescence intensity of thewhole image, I@is the value of I immediately after the filling of all vessels byRho-labeledSL or BSA.The averagehematocrit(HT)of tumormicrovesselswas assumed to be equal to 19% (10). Vand S are the total volume and surfacearea of vessels within the tissue volume covered by the surface image,respectively. Their ratio was estimated by:

@;= n@l 4d.L./@d.2Ln

where d@and 4, are the diameter and the length of the nthvessel, respectively,and M is the total number of vessels on the tumor surface within the image. Themeasurement of vessel diameter in a fluorescence image was corrected by afactor of 0.79 due to light scattering in the tissue (8).

The plasma clearance of BSA was obtained in this study by sampling bloodthrough the carotid artery at different time points after injection of Rho-labeledBSA. The blood samples were then centrifuged, and the plasma was collectedfor measurement of its fluorescence intensity. The plasma pharmacokinetics ofSL was directly obtained from Huang CCa!. (5). The time constant of plasma

clearance (K) was calculated by curve-fitting plasma pharmacokinetics oftracers (Rho-labeled SL and BSA) to an exponential function. The results were9.1X10@ S for BSA and 3.7X10― s for SL.

The in vitro calibration of the system was performed, and a linear correlation between fluorescence intensity measured by photomultiplier and tracerconcentrationwas obtained(r@> 0.998).Mann-WhitneyU test was used tocompare the difference in the effective microvascular permeabilities betweenBSA and SL

Results

The microvessels in normal s.c. tissue have been classified asarterioles, capillaries, postcapillary venules (confluent capillaries),and collecting venules; tumor vessels were not classified in our study(9). The vessel wall was used as a reference structure when describingdistribution of SL in tissue, “intramural―means within the vessel wall,whereas “perivascular―indicates outside and around the vessel wall.

The dominant feature of liposome accumulation in tumors wasperivascular distribution of fluorescent spots (<20 @imin diameter;Fig. Ut). Some of the spots were clusters of extravasated liposomes(Fig. IA), since i.v. injection of Rho-labeled BSA or free Rho resultedin a relatively uniform distribution of fluorescence in tumor interstitium. The rest of the fluorescent spots were fluorescently labeled cellswith dark nuclei at the center (Fig. IA). The appearance of thefluorescently labeled cells suggested the phagocytosis ofliposomes bymacrophages and/or tumor cells. It is also possible that some of thesefluorescent spots were leukocytes extravasated after engulfing liposomes in the blood stream (11, 12). The first appearance of thefluorescent spots could be detected immediately after liposome injec

tion; and the number of spots increased with time. Maximum extravasation was reached in about one day after injection when mostliposomes had been cleared from the plasma, indicated by the reduc

tion of fluorescence intensity in the lumen of vessels. Thereafter, thesize and fluorescence intensity of these spots decreased and could notbe detected at about 1 week after injection. Throughout this timeperiod, most fluorescent spots were located within a distance of 30@imfrom the vessel wall. To simplify the description, we defined the

tumor vessels with perivascular accumulation of liposomes as leakyvessels. The number of leaky vessels varied between 30 and 90% ofthe total vessels at surface among tumors, and their distribution washeterogeneous. Two vascular networks on the surface of a tumor wereobserved to have different patterns of liposome extravasation (Fig.1B), although blood flow was observed in both networks. At the

advancing edge of angiogenesis, extravasated liposomes were mostlylocated near the roots of capillary sprouts, whereas the capillary

(A) sprouts per se showed minimal leakiness 1 day after injection (Fig.1C). Intramural accumulation of SL was observed in less than 10% oftumor vessels, which was less frequent than the number of leakyvessels.

In normal s.c. tissue, extravasation of SL was minimal, and nofluorescent spot was detected in the extravascular compartment duringa 2-week observation. Only the intramural accumulation of fluorescent spots was observed within 5 mm after liposome injection andcould be continuously observed for up to 2 weeks (Fig. 1D). Thesevessels (6 to 25 ,@min diameter) were identified as postcapillary andcollecting venules (9). The fluorescent spots in the vessel wall were2—15 @imin length and 1—2@.amin width and were located adjacent to

the plasma layer. When the entire s.c. tissue in the chamber area wasscanned, about 2% of postcapillary venules and 10% of collectingvenules were labeled with fluorescence; and most labeled vessels haddiameters of 10 to 15 @&m.Fluorescence labeling of the vessel wallcould not be observed after i.v. injection of Rho-labeled BSA, whileinjection of free Rho caused a uniform staining of the wall of allvessels (arterioles, capillaries, and venules). Thus, the punctated stain

ing of postcapillary and collecting venules shown in Fig. 1D was

caused by the Rho-labeled SL. Neither parallel capillaries nor artenoles and large collecting venules (>25 tim) were labeled by liposomes (Fig. 1D). We do not know the mechanism for this preferentialaccumulation, but it is probably related to specific properties ofendothelial cells of these vessels, since macromolecules also showpreferential extravasation from venules (13).

In order to identify the location of liposome accumulation in thevenule wall, we superfused the s.c. tissue with histamine. We foundthat patterns of individual fluorescence spots observed immediately or1 day before superfusion could not be altered by histamine, eventhough histamine treatment caused vessel dilation and leakiness to theSL in s.c. tissue. Histamine-induced vascular leakage was much moresevere than the leakage in tumors (not treated with histamine), asrevealed by the direct observation of liposome accumulation in cxtravascular space and the measurement of total fluorescence intensity(results not shown). In addition, histamine treatment caused an enhanced fluorescence labeling of venules, i.e., nearly all venules werelabeled, and more fluorescent spots were observed in the wall of each

venule.To quantify the leakiness of tumor vessels, we measured the effec

tive permeability of tumor microvessels (P) as shown in Table 1. Wefound that P = 2.0 ±1.6 X 108 cm/s (n = 23) to the SL. This is sixtimes smaller (P < 0.001) than that to BSA (1.2 ±0.5 X iO@ cm/s;n 6). If the fractional volume of tumor vasculature is assumed to be9.2% (8), then we can calculate, from the results in Table 1, the meanvalues of the permeability-surface area product per unit tumor volume. They are 0.32 X i0@ â€and 0.52 X i0@ â€for BSA and

(B)

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UPO5OME DELIVERY IN TUMORS

DCV

C

a

Fig. 1. Extravasation of liposomes in normal and tumor tissue. A, the dominant feature of the liposome accumulation in tumors was perivascular distribution of fluorescent spots(<20 @smin diameter). Some of the spots were fluorescently labeled cells with dark nuclei at the center (arrows); the rest of the fluorescent spots were clusters of the extravasatedliposomes. The photograph was taken 2 days after liposome injection.Bar, 100 @.un.B, two vascular networks on the surface of a tumor had different patterns of liposome extravasation.There was a significant extravasation on the left, whereas insignificant extravasation occurred on the right. Bar, 400 @tm.C, the liposome extravasation at the advancing edge ofangiogenesis was heterogeneous. The vessels grow from bottom to top in the photograph. The extravasated liposomes were mostly located near the roots of capillary sprouts (bottom),while the capillary sproutsperse(top) showed minimal leakiness. Bar, 200 p.m.D, in normal s.c. tissue, only the Intramural accumulation offluorescent spots(arrows)in postcapillaryand collecting venules (6 to 25 p.m in diameter) was observed. Neither parallel capillaries (c) nor arterioles (a) and large collecting venules (v; >25 sun) were labeled by liposomes.Bar, 100 p@rn.

SL, respectively. There was no correlation between tumor size or ageand permeability.

Discussion

In this study, distinct patterns of SL accumulation in normal versustumor tissue were observed. The tumor vessels were leaky to SL withthe permeability about 16% of that to BSA. The distribution of leakyvessels was heterogeneous. The differential accumulation of SL allows us to explore possible mechanisms of liposome extravasationand SL-mediated improvement of drug delivery in tumors.

Intracellular versus Extracellular Accumulation In NormalVessels. Although we have observed the accumulation of SL innormal s.c. vessel walls, the resolution of our technique does not allowus to distinguish intracellular uptake from extracellular accumulation.However, information on intracellular versus extracellular accumulation of SL could be obtained indirectly by the following observations:(a) the pattern of preexisting fluorescent spots could not be changed

by histamine superfusion of s.c. tissue, suggesting that the liposomeswere internalized by cells (endothelial cells and pericytes); (b) histamine can significantly increase the number of pinocytotic vesicles in

endothelial cells of microvessels (14) and enhance fluorescence labeling of venules observed in our study. The comparison of the resultsin these two studies suggests that the fluorescence labeling of the

venule wall can be attributed to endothelial uptake of fluorescentlylabeled liposomes; (c) it is unlikely that accumulation of SL was onthe luminal surface of endothelial cells, since (i) the thickness of mostfluorescent spots was greater than 1 @.tm,which was much larger thanthe diameter (—0.1p@m)of the liposomes and the thickness (<0.1 hun)of the extracellular matrix coated on the luminal surface of endothelialcells and (ii) the shear stress of blood flow would prevent liposomesfrom forming many layers of aggregates on endothelial surface; and(d) it is also unlikely that the intramural accumulation of fluorescentspots is due to leukocyte accumulation between endothelial cells andbasement membrane after they have engulfed liposomes in plasma,because (i) if that was the case, they would be expected to extravasateby releasing enzymes to degrade basement membrane (15) and (ii) wedid not observe any adhesion of fluorescently labeled leukocytes tothe luminal surface of vessel wall or fluorescently labeled cells in theextravascular space during a 2-week intermittent observation. Takentogether, we suggest that the intramural accumulation of SL in s.c.postcapillary and collecting venules is possibly via endocytosis byendothelial cells. This is consistent with the recent observations of thelocalization of colloidal gold-containing liposomes within endotheialcells (6).

Possible Mechanism of Liposome Extravasation in Tumors.There are two bathers for liposome extravasation, the microvascular

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Table 1 Effective microvascular permeability to BSA and SL intumors@rSAa

(l@.um2)Tumor

Age(days)S/I―

X iO@(rm2/rm3)P'@

X lO@(cm/s)PS/V―

X iO@(@_1)MeanBSA

(n =6)13.0203.011.243.51Median12.8213.051.393.66Range10.0-17.618—222.03—3.920.56-1.672.21-4.70SD2.720.720.450.97MeanSL

(n23)18.4243.100.1950.570Median16.8223.030.1550.432Range9.4-39.015-321.93-4.600.002-03680.005-1.68SD8.140.760.1570.453

UPOSOMEDEUVERYIN TUMORS

a Tumor surface area, which reflects the size of tumors.l@@tioof total surfaceareaandvolumeof tumorvessels.

CEffective microvascular permeability.d Product of P and S/V.

labeled leukocytes in plasma observed in a 0.5 XO.5 mm2 image in 1mm cannot account for the significant extravasation of liposomesdetected by the measurement of fluorescence intensity. In addition,nearly no sticking of fluorescently labeled leukocytes was observedon the luminal surface of the vessel wall, a step that is necessary forleukocyte extravasation.

Effective Microvascular Permeability. The effective microvascular permeability is related to the diffusive permeability coefficient,the solute reflection coefficient of microvessels, and the convectiveflux of fluid across the vessel wall (8, 13, 22, 23). For macromoleculesand large particles (e.g., liposomes), the dominant mode of transvascular transport is convection (13) because of the lower diffusioncoefficient. Thus, the value of effective permeability to liposomesreflects primarily convective transport of liposomes across the vesselwall. We expect the effective permeability at the tumor surface to behigher than that in the center, because the transmural pressure difference which governs the convective transport is higher at the surface(24), unless there is a significant difference in the structure of thevessel wall between the center and surface of tumors. Our results onpermeability are qualitatively similar to that of Wu et aL (7). Bothstudies show smaller permeability to SL than to BSA. However, theratio of the permeabilities between SL and BSA is about 16% in ourstudy and 50% in study of Wu et a!. (7). This discrepancy might beexplained by the differences in tumors and liposomes. The mean valueof the microvascular permeability to BSA in the present study wasabout five times smaller than that in the study of Yuan et aL (8) dueto a refinement in the method of analysis.

Improved Drug Delivery in Tumors. SLs have improved druglocalization and efficacy in tumors (1—3).The prolonged plasmahalf-life and lower extravasation in normal tissue are only part of thereason for this improvement. Our observations reveal that once theliposomes extravasate, they form perivascular clusters and do notmove significantly in tumor interstitial space. Thus, the retention ofdrugs in tumors will be increased (up to a few days). As a comparison,when the drug molecules without specific binding (e.g., doxorubicin)are injected alone, they will be quickly cleared from plasma andtissue, resulting in a much shorter period of drug supply in tumors.The results of penvascular accumulation of liposomes in the presentstudy provide the first direct evidence to support our current hypothesis that the liposomes extravasate in tumors after vascularization andform depots of drugs in the perivascular space, which are available toneighboring cells within tumors for up to a few days. This sustainedlocal release of drugs is especially important after the liposomeencapsulated drugs are cleared from plasma. In addition, these resultshave useful implications for the delivery of antiangiogenic agents andimmunoliposomes.

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endothelium and the basement membrane. Previous studies indicatethat liposomes may cross vascular endothelium via open spaces between endothelial cells as well as transcytotic vesicles in tumors (5,6)or activated circulating monocytes/macrophages in the lung (11) andaorta (12). However, the present study suggests that neither vesiculartransport nor leukocyte-mediated extravasation are likely to be themain pathway of transendothelial transport of liposomes in tumors.

The size of SL used in this study (mean diameter, —90nm) iscomparable with or larger than the size of transendothelial vesicles(—50to 70 nm) in both normal (16) and tumor tissue (17). Thus, SLcannot be easily engulfed then shuttled by vesicles or move freelythrough vesicular channels (18) or vesiculo-vacuolar organelles (17).Even though the liposomes can be taken up by large vacuoles (6, 17),these vacuoles are less frequent and may not traverse across cells. Theresults in normal s.c. tissue support this argument, since the liposomesapparently internalized by endothelial cells remain trapped intracellularly for several days, much longer than the normal shuttling time(— 10 mm) of vesicles observed in the studies of macromolecular

transport (16, 18). Since the significant extravasation of SL intumors could be detected immediately after injection in our study,we hypothesize that the major pathway of transendothelial transport in our tumor model is the large gaps between endothelial cells.This hypothesis, however, does not exclude the possibility that asmall fraction of extravasated liposomes is via transendothelialvesicles and/or vacuoles.

The basement membrane is the second barrier for liposome extravasation. Large particles (e.g., colloidal carbon and SL) can accumulatebetween endothelial cells and basement membrane in both normal(19)andtumortissue(5, 20).However,thebasementmembraneisdiscontinuous at certain locations, which allows penetration of largeparticles (19). After crossing vascular endothelium, the Iiposomesmay travel laterally, then cross the basement membrane at discontinuous regions. Therefore, we propose that the basement membranemay not be a major barrier for liposome extravasation. This hypothesis is supported by two observations: (a) in normal s.c. tissue, largenumbers of SL penetrated into interstitial space after the opening ofendothelial junctions by histamine treatment; and (b) perivascularaccumulation of liposomes was the dominant feature of the liposomedistribution in tumors (Fig. IA). The structure of basement membranechanges constantly and depends on the balance between productionand degradation by cells (e.g., endothelial cells, fibroblasts, tumorcells, etc.; Ref. 21). The absence or incompleteness of vascularbasement membrane in tumors may facilitate extravasation and thusincrease the permeability of tumor vessels to liposomes.

We cannot exclude the possibility that some liposomes extravasatedin tumors with leukocytes. However, at most, three fluorescenfly

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UPOSOME DEUVERY IN TUMORS

269—276,1993.11. Poste, 0., Bucana, C., Raz, A, Bugelski, P., Kirsh, R., and Fidler, I. J. Analysis of the

fate of systemically administered liposomes and implications for their use in drugdelivery. Cancer Res., 42: 1412—1422,1982.

12. Hodis, H. N., Amartey, J. IC, Crawford, D. W., Wickham, E., Sharma, R. C., andBlankenhorn, D. H. Relationship of arterial wall uptake of radiolabeled liposomes tothe presence of monocyte/macrophage cells in the hypertensive and atheroscleroticarterial wall. Atherosclerosis, 87: 109—117,1991.

13. Jam, R. K. Transport of molecules across tumor vasculature. Cancer Metastasis Rev.,6: 559—593,1987.

14. Dux, E. and Joó,F. Effects of histamine on brain capillaries. Exp. Brain Res., 47:252—258,1982.

15. Matzner, Y., Bar-Ncr, M., Yahalom, J., Ishai-Michaei, R., Fuks, Z., and Vlodavsky,I. Degradation of heparan sulfate in the subendothelial extracellular matrix by areadily released heparanase from human neutrophils. J. Chin. Invest., 76: 1306—1313,1985.

16. Predescu, D., and Palade, U. E. Plasmalemmal vesicles represent the large poresystem of continuous microvascular endothelium. Am. J. Physiol., 265: H725—H733,1993.

17. Kobn, S., Nagy, J. A., Dvorak, H. F., and Dvorak, A. M. Pathways of macromoleculartracer transport across venules and small veins. Lab. Invest., 67: 596—607,1992.

18. Michel, C. C. The transport of albumin: a critique of the vesicular system intransendothelial transport. Am. Rev. Respir. Dis., 146: 532—536,1992.

19. Majno, 0., and Palade, G. E. Studies on inflammation. 1. The effect of histamineand serotonin on vascular permeability: an electron microscopic study. J. Biophys.Biochem. Cytol., 11: 571—605,1961.

20. Dvorak, H. F., Nagy, J. A., Dvorak, J. T., and Dvorak, A. M. Identification andcharacterization of the blood vessels of solid tumors that are leaky to circulatingmacromolecules. Am. J. PathoL, 133: 95-109, 1988.

21. Bosman, F. T., Havenith, M., and Cleutjens, J. P. M. Basement membranes in cancer.Ultrastruct. Pathol., 8: 291—304,1985.

22. Gerlowski, L E., and Jam, R. K. Microvascular permeability ofnormal and neoplastictissues. Microvasc. Res., 31: 288-305, 1986.

23. Jam, R. K. Physiological resistance to the treatment of solid tumors. In: B. A. Teicher(esis.), Drug Resistance in Oncology, pp. 87—105. New York: Marcel Dekker, Inc.,1993.

24. Boucher, Y., Baxter, L. T., and Jam, R. K. Interstitial pressure gradients in tissueisolated and subcutaneous tumors: implications for therapy. Cancer Rca., 50: 4478—4484, 1990.

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Acknowledgments

We thankJuliaKahnforchamberpreparationsandDr.Yves Boucherforhishelpful comments.

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