JOURNAL OF LIPOSOME RESEARCH, 2(3), 451-454 (1992)

EPILOGUE

STEALTH LIPOSOMES”, NINJA LIPOSOMES, OR CRYPTOSOMES: ARE

THEY REALLY STERICALLY STABILIZED LIPOSOMES?

Leaf Huang Department of Pharmacology

University of Pittsburgh School of Medicine Pittsburgh, PA 15261

When the idea of putting together a Forum on the subject of polymer

and glycan coated liposomes came across my mind, I thought that this would

be an unique opportunity for those of us who argue fiercefully in meetings to

do the same in writing. The idea was well received when I contacted my

colleagues about a year ago. After some minor arm-twisting by phone and FAX, here is the first Forum published in JLR.

As expected, there is some disagreement on the details of how

polyethylene glycol and other glycans reduce the uptake of liposomes by RES’.

The work of Chonn and Cullis (see article in this Forum) clearly show that the

presence of ganglioside G,, or PEG on the liposome surface drastically reduces

the amount of blood proteins coating the liposome surface. Some of these

proteins are undoubtedly the opsonins. The question is: by what mechanism

the polymer and glycans can resist the protein coating of liposomes?

1. Abbreviations: PEG: polyethylene glycol; PEG5000-PE: polyethylene

glycol 5000 conjugated to phosphatidylethanolamine; PI: phosphatidylinositol;

RES: reticuloendothelial system.

45 1

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452 EPILOGUE

The presence of a steric barrier on the PEG-coated liposomes is

demonstrated by us using a liposome agglutination assay (l), and by Needham

et al. by using X-ray diffraction (see article in this Forum). Whether the steric

barrier activity is directly responsible for the prolongation of liposome

circulation, as suggested by several authors of this Forum, has not been adequately demonstrated, although there is a positive correlation between the

two (1). We have shown that at low concentrations of PEG5000-PE

streptavidin can still bind to the biotin groups on the liposome surface, yet it

can not mediate any significant liposome agglutination (2). These results imply

that PEG coated liposomes may show suppressed RES uptake without

exhibiting substantial reduction in the amount of blood protein coating.

Another point about steric barrier mechanism is that not all steric

barriers confer the activity to resist RES uptake of liposomes. Sunamoto et al.

have described the coating of liposomes with polysaccharide such as pullulan

and arnylopectin (3). The molecular weight of these polymers is so large that

the steric barrier activity, although not directly measured, would be greater

than that of PEG5000, yet the polysaccharide coated liposomes do not show any

appreciable activity to prolong the circulation time of liposomes.

The paper by Blume and Cevc (see article in this Forum) has described

another interesting observation, i.e. the importance of functional group on the

tip of the PEG chain. The presence of a carboxyl group seems to result in an

enhanced liposome uptake by the intestines. This surprising observation, if

confirmed, suggests that we can still learn a lot from the basic PEG chemistry,

and from the interactions of PEG with lipid bilayer and the surrounding

biological fluid.

How do glycans resist protein coating on the liposome surface? Both

our work (1) and that of Needham et al. (see article in this Forum) indicate

that G,, only present a weak steric barrier at the liposome surface. It is not

likely that the activity to resist RES uptake can be accounted for solely on the

basis of this weak barrier activity. G,, does not significantly increase the

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EPILOGUE 453

surface hydrophilicity of liposomes as measured by a two-phase partition

method (our unpublished data). On the other hand, chemical modifications on

the C5 and C1 positions of the sialic acid residue of G M 1 indicate that a specific

structure of the residue is required for the activity (unpublished data). Interestingly, the activity of G,, in binding with the cholera toxin has the

similar structural requirement. These results taken together suggest that

specific recognition of GMi by presumably a serum proteids), i.e. a

dysopsonin(s), is a prerequisite step in resisting the RES uptake of liposomes.

Obviously, many pieces of the puzzle are still missing before we can understand

how G M 1 works.

The headgroup of GM, contains five saccharide units. Other glycolipids

of much smaller size also possess similar but weaker activities as that of GM1.

A well known example is PI. Interestingly, Chonn and Cullis have shown that

only PI from plant sources has the activity; PI extracted from the liver does not

(see article in this Forum). Is the fatty acyl chain unsaturation of PI important

for the activity, or, is it some subtle difference in the inositol conformation

which determines the activity? Also interesting is the report by Oku et al. that

glucuronic acid conjugated to a palmityl chain can effectively reduce liposome

uptake by the RES (4). Other glycolipids of plant sources also modify the

liposome’s clearance rate and biodistribution as reviewed by Ghosh and

Bachhawat (5). These relatively small natural and synthetic glycolipids are not

expected to present a strong steric barrier at the liposome surface. It is more

likely that they express the activity by mimicking some structural feature of

GM1 and interacting with the hypothetical dysopsonin(s).

We have purposely avoided or de-emphasized the application, i.e. drug

delivery, side of the story in this Forum, because it can and should be the

subject of a separate Forum. The work in the last few years on the long

circulating liposomes has undoubtedly brought new life into the liposome field.

The author of a recent article in a scientific news journal also recognizes this

fact (6). However, as clearly shown by the authors of this Forum, there are still

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454 EPILOGUE

many unresolved problems. The dust has not settled. The challenge is still

ahead of us.

I wish to thank all authors for their contributions and wisdom on the

organization of the Forum. Particularly, I thank my secretary Linda Spotts for her untiring work of manuscript editing, without which this new experiment

of JLR would not be possible.

REFERENCES

1. Mori, A., Klibanov, A.L., Torchilin, V.P. and Huang, L. 1991. Influence of the steric barrier activity of amphipathic poly(ethyleneglyco1) and ganglioside G,, 011 the circulation time of liposomes and on the target binding of immunoliposomes in vivo. FEBS Lett. 284: 263.

2. Klibanov, A.L., Maruyama, K., Beckerley, AM., Torchilin, V.P. and Huang, E. 1991. Activity of amphipathic poly(ethyleneglyco1) 5000 to prolong the circulation time of liposomes depends on the liposome size and is unfavorable for irmnunoliposome binding to target. Biochim. Biophys. Acta 1062: 142.

3. Sunamoto, J. Iwamoto, K., Masahiro, T., Yuzuriha, T. and Katayama, K. 1983. Polymer coated liposomes for drug delivery to target specific organs. In: "Recent Advances in Drug Delivery Systems". Eds. Anderson, J.M. and Kim, S.W., Plenum Press, New York, pp. 153-162.

4. Glucuronate-modified liposomes with prolonged circulation time. Pharm. Bull. 38: 1663.

N m b a , Y. Sakakibara, T., Masada, M., Ito, F. and Oku, N. 1990. Chem.

5. Ghosh, P. and Bachhawat, B.K. 1980. Grafting of different glycosides on the surface of liposomes and its effect on the tissue distribution of '251-labelled a-globulin encapsulated in liposomes. Biochim. Biophys. Acta 632: 562.

6. Hamood, J.L. 1992. Understanding liposomal properties to aid their clinical usage. Trends In Biochemical Sciences. 17: 203.

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