Volume of distribution and transcapillary passage of small unilamellar vesicles

Life Sciences, Vol. 31, pp. 949-955 Printed in the U.S.A.

Pergamon Press

VOLUME OF DISTRIBUTION AND TRANSCAPILLARY PASSAGE OF SMALL UNILAMELLAR VESICLES Karl J. Hwang, Kuen-Fai Steven Luk and Paul L. Beaumier D e p a r t m e n t of Pharmaceutics, School of Pharmacy, BG-20 University of Washington, Seattle, WA 98195 (Received in final form June 18, 1982)

Summary This study investigated the biodistribution of bovine brain sphingomyelin (SM)/cholesterol (CH) (2/I; M/M) small unilamelIar vesicles (SUV) in mice, addressing specifically the volume of distribution and transcapilIary passage of the SUV. The complex of n i t r i l o t r i a c e t i c acid with I n - l l l or Ga-67 ions was encapsulated in the SUV as the radioactive marker for various studies. The structural integrity of liposomes in vitro and in vivo was monitored by the technique of gamma ray perturbed angular correlation. Our data suggested that i n i t i a l l y the SM/CH SUV remained within the vascular system and occupied a volume of distribution approximately 1.28 times larger than that of erythrocytes in the vascular system of mice. However, our data also indicated that with time the SM/CH SUV could get out of the vascular system of mice and were taken up by surrounding tissues over a period of 24 hours. Liposomes have been proposed to be useful drug carriers in vivo. Therapeutic agents entrapped in liposomes have been applied to model systems as diverse as heavy metal detoxification (1), t r e a t m e n t of parasitic disease (2-4), and cancer chemotherapy (5). The various uses of liposomes to deliver pharmacologically active agents for therapeutic applications have been reviewed (6, 7). Intravenously administered liposomes are taken up avidly by the reticuloendothelial system (RES) (8, 9). Therefore, liposome-encapsulated drugs are found to be very e f f e c t i v e in t r e a t i n g diseases, such as visceral leishmaniasis (24), which occur in the RES. In order to deliver drugs to tissues other than the RES using liposomes, two import a n t c r i t e r i a must be met in the design of liposomes. First, liposomes must be able to evade rapid uptake by the RES and stay in the circulating blood for an extended period of time to allow direction of the liposome to the desired tissues. Second, if homing molecules are attached to the liposomal surface, there should be sufficient i n t e r a c t i o n between the site-specific molecules (carbohydrates, or antibodies) on the surface of liposomes and receptors or antigens on the surface of the targeted ceils. This suggests that in order for liposomes to be e f f e c t i v e in delivering drugs to target tissues after intravenous administration, they must be able to pass out of the vascular system, allowing suitable c o n t a c t or i n t e r a c t i o n with the t a r g e t ceils. There are at ]east two ways to study whether and to what extent liposomes can pass out of the vascular system. The f i r s t approach, direct measurement of the amount of intact liposomes present in the extravascular space, requires analytical methods which differentiate the intact liposomes present in the extravascular space from the liposomal marker which is released from degraded liposomes and subsequently transported to the extravascular space. An alternative approach is to study the time course of the biodistribution of the liposomes which are known to remain intact in the circulating blood. If the possibility that liposomes are bound and/or trapped transiently by blood vessels can 0024-3205/82/100949-07503.00/0 Copyright (c) 1982 Pergamon Press Ltd.

950

Fate of Small Unilamellar Vesicles In Viuo

Vol. 31, No. i0, 1982

be excluded, a gradual increase in the uptake of such liposomes in organs or tissues with time would suggest that the i n t a c t liposomes pass out of the vascular system and are taken up by surrounding tissues. Recent studies on the bovine brain sphingomyelin (SM)/cholesterol (CH) small unilamellar vesicles (SUV) from our laboratory (I0) and others (I I) indicated that this type of liposome remains intact and retains entrapped substances in the circulating blood of mice with an unusually long h a l f - l i f e of 16-17 hrs. The present report addresses the question of whether or not the long-lived SM/CH (2/I; M/M) SUV can pass out of the vascular system of mice, using the second approach mentioned oin the previous paragraph. Our results indicate that SM/CH (2/I; M/M) SUV (about 200 A in diameter) distribute in a volume 1.28 times the volume of distribution of red blood cells and that the SUV can pass out of the vascular system in vivo. Materials and Methods All lipids were purchased from Sigma Chemical Company. Oxine and n i t r i l o t r i a c e t i c acid (NTA) were obtained from Aldrich Chemical Co. Gallium-67 citrate and i n d i u m - I l l chloride were purchased from Medi+Physics. The resins, A G I X - 8 (chloride form) and Chelex 100, were obtained from BioRad. Sephadex G-50 and Sepharose 4B were purchased from Pharmacia. The purification of In-I I I chloride and isolation of fresh defibrinated rabbit serum were carried out as described previously (12). Ga-67 chloride was prepared from Ga-67citrate by chromatography on an anion exchange column. The gallium citrate was converted to Ga-67 chloride by adding an appropriate volume of 6N HCJ producing a final concentration of 2N HCI, before applying to a Bio-Rad A G I X - 8 column, 0.7x10 cm, equilibrated with 2N HCI. The citrate was eluted by two bed volumes of 2N HCI, and the Ga-67 chloride was isolated by a step gradient of one bed volume of 0.1N HCI. The fractions were collected in Nalgene beakers and taken to dryness under heat lamps. The A G I X - 8 resin (phosphate form) was prepared by cycling the commercially available chloride form of the resin in sodium hydroxide, deionized water and IN phosphoric acid until no chloride ions were present in the wash solution, as indicated by the silver nitrate test for chloride ion. The anion exchange resin was then equilibrated with 0.106M sodium phosphate, pH 7.4. Small unilamellar vesicles (SUV) were prepared by sonicating the dried thin film of lipids and ImM NTA in an appropriate isotonic buffered solution at the desired pH in a Branson 350W sonicator with a titanium microtip at the setting of 1.5 as described previously (I0). Titanium fragments and any multilamellar liposomes were removed by centrifuging at 160,000 g for l hr. The average size of the liposomes was estimated to be 187-+42 A from negative-stain electron micrographs of the liposomes using potassium phosphotungstate as the stain. Untrapped materials were removed by passage of the liposomes over a Sephadex G-50 column, 0.8 x 35 cm, in 0.9% NaCI, 5mM sodium a c e t a t e , pH 5.5. To encapsulate i n d i u m - I l l or gallium-67, I n - l l l or Ga-67 was delivered to the encapsulated lmM NTA by means of a mobile ionophore, 8-hydroxyquino!ine as described previously (i0, 13). The e x t e n t of the leakage of the entrapped cations in the presence or absence of serum was determined by chromatographing on Sepharose 4B and/or by the technique of gamma ray perturbed angular correlation (PAC) as described previously (12, 13). For in vivo studies, liposomes were injected via the tail vein to BALB/c mice weighing about 25 g. The injected dose was estimated by weighing the syringe before and after injection and multiplying by the specific activity of the preparation. The mice were sacrificed at various times post-injection by cervical dislocation and immediate decapitation. The various organs and tissues of the mouse were isolated, rinsed with saline, blotted, weighed, and counted in a gamma counter. Results are expressed as percentage of the injected dose. In dual labeling experiments, the crossover was estimated by appropriate standards and corrected by a computer program. In all tissue distribution

Fate of Small Unilamellar Vesicles In Viuo

Vol. 31, No. i0, 1982

951

studies, the radioactivity due to the blood background in each sample was corrected. The correction was estimated from the distribution of red blood cells labeled with oxine-ln-I l l (10, l#). The volume of distribution of red blood cells and the SM/CH (2/I; M/M) SUV, expressed as a percentage of the body weight, was determined according to the procedures described previously (I 0). RESULTS

The average volume of distribution of red blood cells of a mouse was found to be g.0~0.196 (n=6) of the total body weight, using fresh mouse erythrocytes labeled with In111 using In-I 1 l-g-hydroxyquinoline. This value agrees closely with previously reported values (17). Figure I depicts the percentage of the total administered dose of SM/CH (2/1; M/M) SUV as estimated from the radioactivity of the liposome-encapsulated I n - I l l present in the vascular system of mice (taking the blood volume, VRBC, as 8.096 of the total body weight) at various periods post-intravenous administration of liposomes. The volume of distribution of the liposomes was found to be 1.2g times VRBC as calculated from the recovery of the administered dose extrapolated to time zero in Figure 1. It is interesting to note that a similar recovery of 7096 of the total injected dose of egg phosphatidylcholine (PC)/CH SUV in the blood at 3 min post administration has also been reported by Kirby and Gregoriadis (16). The time course of the biodistribution of liposomes is shown in Table I. I0090-

80"~ 7 0 o 60o3

13._ -3 50c]

,,, 4 0 -

F(O 1,1

-~

30-

z

2O

I 0

'

I

'

I

J I

'

I

'

1

40 80 120 160200 T I M E , MIN FIG. l.

Clearance of SM/CH (2/1; M/M) SUV from the blood of mice. The percentage of injected liposomes in the blood was calculated from the specific a c t i v i t y of the liposome-entrapped I n - I l l in the blood and an average blood volume of 8.096 of the body weight. The number of animals for each point were 3, g, 3, 6, and 10 at I, 15, 30, 60, and 180 minutes post-injection, respectively. The dotted line is the extrapolation of the straight line connecting 15, 309 60, and 180 minutes to time zero. The a c t i v i t y at time zero was estimated to be 7g% of the injected liposomes. To investigate whether the recovery of g0% of the injected liposomes in the blood at l minute post injection was a result of a saturation phenomenon, pulse-chase experiments were performed. Three typical results of the pulse-chase experiments are shown in Table If. At time zero, SM/CH (2/I; M/M) SUV entrapping Ga-67 were injected into each mouse.

Fate of Small Unilamellar Vesicles fn V i v o

952

Vol. 31, No. i0, 1982

After 15 min, each mouse received a second intravenous injection of the same liposomes entrapping I n - l l l . The animals were sacrificed at either 1 min or 15 min after the second injection of liposomes. In all cases, only a maximum of 8096 of the second injected dose of liposomes could be found in the blood. This suggested that a saturation phenomenon was not likely since a 100% recovery of the second injected dose of liposomes (entrapping a different isotope) was not obtained. TABLE I Time Course of Biodistribution of Injected Liposomal Radioactivity in Mice*

Percentage (_+SD) of Administered Dose at Various Times After Injection Tissue

l Min (N=3)

15 Min (N=8)

1 Hr (N=6)

3 Hr (N=I0)

Blood

30.0(0.2)

64.8(1.9)

15.7(7.2)

2.8(0.3) 1.0(0.0) 0.0(0.0) 0.0(0.0) 3.6(1.9) 0.7(0.0) 0.4(0.3) 1.7(0.7) 1.0(0.6) 2.0(0.4) 6.5(1.2) 0.2(0.1) 0.1(0.1)

75.1(2.1) 4.9(0.2)

72.5(2.3)

Liver

5.4(1.0) 1.3(0.4) 0.0(0.0) 0.4(0.4) 1.7(9.7) 1.8(0.9) 0.5(0.3) 2.9(0.3) 2.0(0.9) 3.1(0.7) 8.0(3.1) 0.2(0.1) 0.1(0.1)

7.3(1.2) 1.5(0.2) 0.3(0.2) 0.6(0.2) 1.8(1.7) 3.0(0.3) 0.5(0.1) 4.2(0.9) 1.9(0.5) 2.9(0.3) 10.8(0.9) 0.i(0.1) 0.3(0.0)

44.3(8.0) 2.1(0.6) 1.8(0.1) 0.4(0.3) 1.0(0.4) 5.7(1.2) 1.2(0.6) 9.3(2.7) 2.2(0.5) 4.9(0.8) 10.7(2.3) 0.1(0.0) 0.6(0.1)

Kidney

Spleen Heart Lung Intestine Fat Skin Tail Legs Carcass Brain Stomach

1.0(0.I) 0.0(0.0) 0.4(0.1) 2.3(0.6) 1.4(0.2) 0.3(0.1) 2.4(1.0) 2.3(0.3) 2.0(0.2) 8.5(1.5) 0.2(0.0) 0.1(0.0)

23 Hr (N=6)

*The liposomes were SM/CH (2/I; M/M) SUV entrapping I n - I l l , and N is the number of animals. The lipid dose administered in I00 ul ranged from 35 to 75 ug total lipid per g mouse body weight. Since In-I I I ions bind t i g h t l y to tissues at the site of the destruction of liposomes and do not redistribute readily to other tissues, only about 0.5% of the injected dose of I n - I l l was excreted in a 23-hr period, resulting in a nearly complete recovery of the label after 23 hr. The extent of the leakage of In-I 11 from liposomes was estimated by the technique of gamma ray perturbed angular correlation (PAC) (I0, 129 13). The strategy adopted for measuring the leakage of I n - I l l from liposomes by PAC was based on the measurement of the tumbling rate of In-I 11 in solution. When the liposomes are intact, the encapsulated In-I I I - N T A complex will tumble rapidly as a small molecular species, giving a characteristically high perturbation factor~, of 0.59!0.02. On the other hand, when the structural integrity of a liposome is perturbed, by serum components for example, the entrapped I n - I l l will leak out and become bound to serum proteins, primarily transferrin (17). The serum-bound I n - I l l will tumble slowly as a macromolecule, giving a characteristically low of about 0.15. In all our in vitro leakage tests, the <022(0o) > of the I n - i l l entrapped in SM/CH (2/I; M/M) SUV remained unchanged at 0.59_+0.02 even after 24 hr incubation with 5096-90% rabbit serum at 37°C, indicating completely intact liposomes. Furthermore, the blood samples isolated from the mice sacrificed at I min throughout the testing period to 24 hr post-injection had a value of 0.5~0.02, suggesting that all the I n - I l l in blood was encapsulated in intact liposomes.

Fate of Small Unilamellar Vesicles In YiOo

Vol. 31, No. I0, 1982

953

TABLE II Biodistribution of Two Successive Injections of Liposomes in P u ls e - C h a s e Experiments*

P e r c e n t a g e of Administered Dose at Various Times A f t er the First Liposomal Injection ( G a - 6 7 ) and the Second Liposomal Injection ( I n - I l l ) to the Same Mouse

N=I

Blood Liver Kidney Spleen Heart Lung Intestine Fat Skin Tail Legs Carcass Brain Stomach

N=I

i Min In-Ill

16 Min Ga-67

15 Min In-lll

79.9 3.0 1,0 0.0 0.0 2.5 0.7 0,6 1.3 1.4 2.2 7.2 0.1 0, 1

80.1 3.0 0.9 0,0 0.0 2.5 0.8 0.7 1.1 1.4 2,0 7.2 0.2 0.1

80.3 3.6 1.2 0,0 0.0 0.8 1.6 0,2 1.6 2.8 1.4 5.9 0,2 0.2

30 Min Ga-67

15 Min In-Ill

76.3 4.6 1.0 0.0 0,0 1.1 2.0 0.2 2.7 2,8 2.1 6 .7 0.2 0 .3

77.8 3.2 0 .9 0 .0 0.4 1.1 1.2 0.3 3.0 2.6 1.5 7.7 0,2 0.2

N=I 30 Min Ga-67

73.9 4.8 0.7 0.1 0.4 1.2 1.5 0 .4 3.2 2.8 2 .0 8.7 0.2 0 .2

*The liposomes used were SM/CH (2/I; M/M) SUV entrapping either In-I I I or Ga-67. Each mouse received two bolus doses of liposomes 15 minutes apart. The first dose was SUV entrapping Ga-67 and the second dose was SUV entrapping In-I I I. DISCUSSION

The present study indicated that the SM/CH (2/I; M/M) SUV remained intact in the circulating blood and were cleared from the blood slowly. The volume of distribution of the SM/CH SUV was found to be larger than that of red blood cells, There are several possible explanations for this observation. First, it is possible that 8096 of the intravenously administered liposomes become rapidly coated with plasma protein and remain in the blood, whereas the other 2096 are only partially protein-coated and are rapidly cleared from the blood during the first minute post-administration. This is unlikely since we found that over a wide lipid dose range, 6-80 jug total lipid/g mouse body weight (data not shown)) only 70-80% of the administered dose is consistently recovered in the blood (taken as VRBC, g.096 of the body weight) during the first 15 rain post-injection. At a low lipid dose one might expect to observe) according to this interpretation) complete protein coating of the administered liposomes and 10096 recovery in the blood at early time points. Second, viewing the passage of SUV across the capillary wall as a concentration-dependent rate process, the liposomal lipid dose should directly affect the rate of liposome egress into the extravascular space during the f i r s t minute post-injection. However) the above mentioned liposome dose argument discounts this interpretation. Third) the possibility that the removal of 2096 of the second pulse of SUV from blood is due to a rapid regeneration of the binding sites (or receptors) which are responsible for the removal of 2096 of the f i r s t pulse of SUV is not likely since) if this were the case) one would expect a rapid removal of another 2096 or more of the injected liposomes from the blood during the period from 15

954

Fate of Small Unilamellar Vesicles In Viuo

Vol. 31, No. i0, 1982

rain to l hr post-injection. However, this is not the case as seen in Table I. Fourth, it is possible that 2096 of the administered SM/CH SUV distribute immediately into an accessible region of the extravascular space within the first minute post-injection. This percentage may represent a segment of the liposome preparation which is small enough to travel rapidly to the extravascular space. Although this interpretation is unusual, it cannot be ruled out. The most likely possibility is that i n i t i a l l y the SM/CH SUV have a larger volume of distribution than red blood cells within the vascular system, which is reasonable since these SM/CH SUV have a diameter approximately 250-fold smaller than erythrocytes. If this is the case, one might anticipate that after the injected liposomes distribute and equilibrate throughout the vascular system, the biodistribution of the SM/CH SUV would remain relatively invariant as long as the liposomes remain intact in the blood circulation. The PAC results clearly indicated that the SM/CH SUV do indeed remain intact in the circulating blood throughout the testing period of 24 hours. However, the results listed in Table I indicate that the biodistribution of the SM/CH SUV changes gradually with time. As shown in Table [, there is a gradual accumulation of liposomal radioactivity in many tissues (eg, skin and intestine), indicating that in addition to the RES system, other tissues participate in the uptake of liposomes. There are at least two possible explanations which may account for the timedependent accumulation of liposomes in some tissues listed in Table I. First, SM/CH SUV bind to the vascular wall rather than distributing to extravascular tissue sites. Second, intact SM/CH SUV pass out of the vascular system and interact with surrounding tissues. If the first explanation were true, one would expect extensive uptake of liposomes at 23 hours post-injection in highly vascularized tissues, such as lung. This was not found to be the case (Table l). Furthermore, we have shown previously that the SM/CH SUV taken up by liver are degraded rapidly in vivo (I0). Thus, if liposomes were bound to vascular walls and degraded in situ, the release of some liposome-entrapped I n - I l l into the blood circulation would be expected. It is known that transferrin-bound [ n - l l I remains in the blood circulation for an extended period of time (lg). The result of the PAC study of the blood samples clearly indicated that no I n - l l l was released or bound to serum proteins during the course of the experiment. Therefore, the most plausible explanation of the gradual accumulation of liposomal radioactivity in certain tissues with time is that intact SM/CH SUV do pass out of the vascular system and gradually accumulate in these tissues, skin and intestine, for example. However9 as shown in Table l, the extent of the transfer of the SM/CH SUV across capillaries in different tissues is not necessarily the same. This is probably related to the anatomic difference of the intrinsic structure of various classes of capillaries as recently pointed out by Poste et al (19). It is interesting to note that the lack of gradual accumulation of the SM/CH SUV in the lung (Table I) is quite consistent with the observation made by Poste et al (19) who used an entirely different approach to show the inability of phosphatidylserine and phosphatidylcholine SUV to pass out of the capillaries of the lung. It is well known that proteins leak through capillary walls and return to the circulating blood via the lymphatics (20). The passage of intravenously injected solid spherical particles (radius 300-700A) of methylmetacrylate across the capillary wall as early as I0-20 rain post-injection has also been documented (21). Undoubtedly, both the size and the deformability of the proteins or particles can affect the intra-and extravascular distribution. The SM/CH (2/l; M/M) SUV are smaller than the methylmetacrylate spheres. It is thus likely that the mechanism that governs the passage of proteins or particles through vascular walls into the extravascular spaces also operates in the case of liposomes. In conclusion, our results indicate that the SM/CH (2/I; M/M) SUV of about 200 A in diameter have a volume of distribution of approximately 1.28 times larger than that of the erythrocytes in mice, and that the 5UV can pass out of the vascular system of mice slowly.

Vol. 31, No. i0, 1982

Fate of Small Unilamellar Vesicles In Vivo

955

ACK NOW LEDG EM ENTS This work was supported in part by Contract No. DAMD 17-78-C-80#9 from the U.S. Army Medical Research and Development Command, American Heart Association of Washington Grants-in-Aid 78-W-515, PHS grants AM 170#7, AM 25608, and NSF Grant PCM 81-05151. PLB is an NIH predoctoral trainee (CA-09081). We are grateful to Dr. 3ohn H. Wiessner for valuable assistance in preparing electron micrographs.

REFERENCES 1. 2.

Y.E. RAHMAN, M.W. ROSENTHAL AND E.A. CERNY, Science 180 300-302 (1973). C.R. ALVING, E.A. STECK, W.L. CHAPMAN, 3R., V.B. WARTS, L.D. HENRICKS, G.M. SWARTZ, 3R. and W.L. HANSON, Proc. Natl. Acad. Sci. USA 75 2959-2963 (1978). 3. C.D.V. BLACK, G.3. WATSON and R.3. WARD, Trans. R. Soc. Top. Med. Hyg. 71 550-552 (1977). #. R.R.C. NEW, M.L. CHANCE, S.C. THOMAS and W. PETERS, 272 55-56 (1978). 5. G. GREGORIADIS, C.P. SWAIN, E.3. WILLS and A.S. TAVILL, Lancet i 1313-1316 (1974). 6. D. PAPAHAD3OPOULOS, Ann. Reports in Med. Chem. 14 250-260 (1979). 7. G. GREGORIADIS, in: Drug Carriers in Biology and Medicine, G. Gregoriadis, ed., pp. 287-341, Academic Press, London, (1979). 8. G. GREGORIADIS and B.E. RYMAN, Eur. 3. Biochem. 24 485-491 (1972). 9. E. WISSE and G. GREGORIADIS, 3. Reticuloendothel. So"~. 18 109 (1975). 10. K.3. HWANG, K.F.S. LUK, and P.L. BEAUMIER, Proc. Natl. Acad. Sci. USA 77 #030-#03# (1980). 11. G. GREGORIADIS and 3. SENIOR, FEBS Lett. 119 #3-#6 (1980). 12. K.3. HWANG and M.R. MAUK, Proc. Natl. Acad. Sci. USA 7# ##91-##95 (1977). 13. K.3. HWANG, 3. Nuclear Med. 19 1162-1170 (1978). 114. M.3. WELCH, M.L. THAKUR, R.E. COLEMAN) M. PATEL, B.A. SIEGEL and M.M. TER-POGOSSIAN, 3. Nucl. Med. 18 558-562 (1977). 15. B.M. MITRUKA, H.M. RAWUSLEY and B.V. VODEHRA, in: Clinicalt Biochemical~ and Hematological Reference Values in Normal Experimental Animals , p. 6, Masson, New York (1977). 16. C. KIRBY and G. GREGORIADIS, Life Sci. 27, 2223-2230 (1980). 17. F. HOSAIN, P.A. MCINTYRE, K. POULOSE, H.S. STERN and H.N. WAGNER, Clin. Chim. Acta 2# 69-75 (1969). 18. D.A. GOODWIN, M.W. SUNDBERG, C.L. DIAMANTI and MEARES, in: Radiopharmaceuticals, G. Subramanian, B.A. Rhodes) 3.F. Cooper, and V.3. Sodd, eds., pp. 80101, The Society of Nuclear Medicine, Inc., New York (1975). 19. G. POSTE, C. BUCANA, A. RAZ, P. BUGELSKI, R. KIRSH and 1.3. FIDLE, Cancer Res. #2 1#12-1#22 (1982). 20. R.M. NAKAMURA, H.L. SPIEGELBERG, S. LEE and W.O. WEIGLE, 3. Immun. 100 376-383 (1968). 21. G. GROTTE, L. 3UHLIN and N. SANDBERG, Acta Physiol. Scand. 50 287-293 (1960).