Modified Liposome Formulations for Cytosolic Delivery of

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Modified Liposome Formulations for Cytosolic Delivery of Macromolecules Downloaded by UNIV OF CALIFORNIA SAN DIEGO on July 25, 2016 | http://pubs.acs.org Publication Date: May 15, 2000 | doi: 10.1021/bk-2000-0752.ch018

Kyung-Dall Lee and Gretchen M. Larson Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109-1065

Powerful therapeutic agents such as polypeptide-based drugs (1), antisense oligonucleotides (2,3), catalytic RNAs (4,5), and plasmid genes (6-8) represent the next generation of pharmaceuticals, but are limited thus far in their efficacy in vivo due to the difficulty in delivering them to the cytosolic space of target cells. The notion that these agents will exert their efficacy only with good delivery strategies that can place these macromolecules into the proper subcellular compartment of target cells is becoming more appreciated in academia, biotechnology, and pharmaceutical industries. Generally, the cytosolic space and the nucleus, which are topologically connected, are the desired target sites for therapeutic effectiveness of macromolecular drugs. Remarkable advances of biomedical research in recent years have identified numerous new targets and novel ammunitions to combat a variety of diseases, including cancer and AIDS; however, the weapons to deliver these potent agents to their targets at therapeutically effective concentrations in vivo are still lacking (9). Their intrinsic membrane impermeability is due to large molecular size or polyanionic characteristics. Such agents cannot cross the membrane barriers of cells, and thus, have limited access to the cytosol and nucleus. The development of a delivery strategy capable of specifically targeting and transporting these macromolecules across cell membranes into the appropriate subcellular compartments will dictate the potential for these drugs to achieve maximum efficacy. We intend to primarily focus on liposomal drug delivery systems and their evolution from conventional formulations to next generation delivery vehicles, which can provide solutions to the problems of large molecular weight therapeutic agent delivery. The mechanism of liposome interaction with cells will be briefly examined in order to illustrate the limitations and unresolved issues associated with liposomal delivery system. Several recent approaches to overcome these limitations, including the effort in our laboratory, will then be discussed.

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© 2000 American Chemical Society

Park and Mrsny; Controlled Drug Delivery ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

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Background From its inception in the sixties, liposome-based drug delivery research has survived the tortuous path common to many promising drug delivery schemes, and en route has seen significant transformation (10). While conceptually attractive, the dream of using liposomes made from naturally occurring lipids to serve as general drug carriers that can circulate much like red blood cells was quickly dismissed. Researchers soon discovered poor pharmacokinetic properties and rapid uptake of conventional liposomes by phagocytic cells in the reticuloendothelial system. Thus, formulation of sterically stabilized liposomes that show prolonged circulation in plasma was a long-sought solution to a critical obstacle (11-13). After many trials and failures, researchers in the liposome field recognized that the uptake of liposomes by macrophages can be drastically modulated or reduced by inclusion of specific lipids such as ganglioside GMI, phosphatidylinositol, or a synthetic lipid, P E G - P E , which has polyethyleneglycol ( P E G ) attached to the headgroup of phosphatidylethanolamine (PE). The exact mechanism by which incorporation of one of these lipids induces longer circulation times is not entirely clear, although reduced binding of proteins, such as opsonizing plasma proteins or macrophage cell surface proteins, is implicated (14,15). Due to the uptake by their natural target, macrophages of the reticuloendothelial system, efforts to target liposomes to other cell types failed until the advent of these long-circulating, sterically stabilized liposomes. The endocytic uptake of conventional liposomes by macrophages is so rapid that a targeting motif on the liposome surface has little opportunity to interact with the receptors on target cells, decreasing its potential effectiveness. The resurrected idea of targeting liposomes to specific cell types through increased plasma circulation and specific ligand-receptor interaction motivated many investigations of conjugated targeting motifs on longcirculating, sterically stabilized liposomes reporting increased efficiency of delivery to specific cells (16). We will not expand much further on this aspect, and refer to other existing reviews (13,17).

Interaction of Liposomes with Cells: Membranes as a Barrier While targeting at the cellular or tissue level is clearly important, membraneimpermeant macromolecules cannot be effective without cytosolic delivery if their sites of action lie in the cytosol. Normal cellular interaction with its outside environment requires the cell to endocytose or phagocytose exogenous molecules or fluid into small, membrane-bounded vesicles called endocytic compartments. While this allows an effective reduction of the extracellular volume that cells must process, the endosomal membranes act as a physical barrier between the outside and the inside of cells. Thus the cytosol is topologically remote from both the cell exterior and the lumenal space of the endosomal compartment. Although they appear to be within the cell, molecules internalized into the lumen of these endocytic compartments



186 technically still remain extracellular. Once the targeted liposomal vehicles transport the drugs to spécifie cells, most cells internalize them into these endocytic compartments. Membrane-impermeant macromolecules, however, remain trapped inside the endocytic pathway and are eventually delivered to the lysosomal compartments for degradation by hydrolases. Thus, even complete, specific delivery of drugs to the target cells has little therapeutic value if the majority of drug is routed to the lysosomal compartment and degraded. Encapsulation of these macromolecules inside liposomes, either conventional or sterically stabilized, renders little advantage to favor their delivery into cytosol. Since the seminal work by Straubinger et al. demonstrating that liposomes are internalized by cells through the normal endocytic route, it has been well-established that liposomes and their contents are delivered to endosomes (18). These investigators prepared liposomes containing colloidal gold particles to follow the liposome's fate by electron microscopy. Their results definitively showed that the frequency of liposome fusion with cellular membranes, if it exists, is very low, thus liposomal cytosolic delivery is still minimal. Subsequent reports using the pyranine dye, HPTS, provided additional supporting evidence (19-21). Later work using the same techniques to follow PEG-PE-containing, sterically stabilized immunoliposomes also suggested that neutral or anionic liposomes in general have the tendency to be taken up by cells into endocytic compartments regardless of their binding mechanism, nonspecific or ligand-receptor-mediated (16). An exception to these general findings is that direct destabilization or fusion with cellular membranes occurs quite extensively when liposomes are made of non-natural, cationic lipids (22); however, current research in this area is beyond the scope of this article. Yet, scattered reports suggest that we cannot entirely dismiss the possibility that a fraction of non-cationic liposomes can deliver their contents into the cytosol of cells under certain conditions and predominantly in complex in vivo cases (23,24). However, delivery in the range that is useful for most therapeutic purposes clearly requires greater efficiency and predictability of cytosolic delivery of liposomal contents. Given such dogmatic generalizations, one might wonder how some pharmacological or therapeutic effects are observed with macromolecular drugs administered without special delivery systems or in conventional liposomal formulations. The cell biological mechanism of this phenomenon is not well understood, although it is speculated that intrinsic defects in the membranes' barrier function along the endocytic routes cause this limited leakage. If a minuscule amount of internalized material did leak out into the cytosol, a pharmacological effect could be achieved since these drugs are extremely potent once in the cytosol. Investigations addressing these fundamental questions are important and potentially beneficial for logical design of cytosolic delivery systems. Delivery via such mechanisms is far from the levels of predictability and effectiveness ideal for pharmaceutical applications, thus, we return our focus to the progress of liposomal formulations toward this goal.


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Strategies for Cytosolic Delivery: Modified Liposome Formulations Several approaches have been taken to reroute the cargo of endocytosed liposomes to the cytosolic space and away from the lysosomal compartment. One prominent strategy is to confer upon the liposomes a mechanism to overcome the membrane barrier of endocytic compartments. Temporarily breaching the endosomal membranes or inducing direct fusion between the internalized liposomal membrane and the endosomal membrane would allow the escape of endocytosed liposomal contents into the cytosolic space before they reach the highly degradative lysosomal compartments. If one envisions using targeted sterically stabilized liposomes which are designed to be delivered into the endocytic compartments of intended cells, such an approach seems particularly valid. The most common paradigm for intracellular delivery has been to mimic viruses, which can efficiently introduce their contents into the cytosol of specific target cells. Viruses often achieve cell specificity by utilizing cell surface proteins as receptors for viral envelope proteins. Once bound to the cell surface, some viruses promote direct fusion between virus membrane and cell plasma membrane. Others are internalized into the endocytic compartment and trigger membrane fusion between the virus and endosome upon acidification of the endocytic compartment (25,26). Both types utilize the activity of the so called "fusion protein", which has the ability to coalesce two tightly apposed membranes. Two main approaches have been proposed and tested in attempting to design liposomes that behave like viruses. One approach is the use of "pH-sensitive" or "acid-labile" liposomes (27-29). The other is incorporation of the function of the fusion protein into liposomal membranes. The former is achieved by incorporating into a lipid bilayer, made primarily of PE, lipids which have a protonatable moiety with a pKa near the acidic environment of endosomes. PE alone cannot form stable bilayers unless additional anionic lipid species are present in the bilayer. When the negative charge is neutralized upon protonation, the PE-containing bilayer collapses, which presumably results in fusion with adjacent membranes, as suggested by several in vitro liposome-based studies (30). However, no definitive experiment has been designed to test the events inside endosomes in vivo. Indirect evidence supports that these pH-sensitive liposomes induce a certain extent of leakage of endosomes, partly through this fusion event. Several experiments using antigen, fluorescent dye, toxin, or reporter genes show that the cytosolic delivery with pH-sensitive liposomes is greater than that with pH-insensitive liposomal formulations (31-33). However, the reported efficiency of cytosolic delivery indicates less frequent direct fusion between liposomal and endosomal membranes than the design hypothesized. Reconstituting the whole viral fusion protein into liposomes to generate proteoliposomes or isolating the causative segment of fusion protein for incorporation into liposomes have been dominant methods for the second approach. Several delivery systems, particularly for oligonucleotides and plasmid genes, have been designed utilizing virus-derived fusion peptides or whole virus (34,35). In some instances, a viral envelope glycoprotein has been reconstituted into liposomes to


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make artificial virus-like particles, called "virosomes", with a hollow aqueous compartment that can accommodate drugs (36). Similar formulations have been also generated by fusing liposomes with viruses. Viruses that have been exploited so far include influenza virus predominantly, as well as Adeno and Sendai viruses (37,38). While most of these formulations augment cytosolic delivery via liposomes, their efficiency as delivery systems has not been at the level currently sought by the drug delivery field. Moreover, methods that use whole virus or reconstitution of viral membrane proteins are technically problematic for use in pharmaceutical formulations.

A New Delivery Strategy inspired from Intracellular Bacteria A new paradigm that has recently been exploited is the cytosolic delivery of macromolecules by mimicking the escape of facultative intracellular bacteria from endocytic compartments into the cytosol. Parasitic bacteria or protozoa that survive by invading and multiplying within the cytoplasm of host cells must have the ability to escape from the endocytic vacuoles. By secreting specialized proteins, Listeria, Shigella, and Trypanosoma cruzi can effectively disrupt endosomal membranes (3941). A different approach to macromolecular delivery and specifically gene delivery relies on the use of bacterial vectors to transfer exogenous genes, exploiting the cell biology of bacterial escape from endosomes (42). Facultative intracellular bacteria including Shigella and Listeria have been utilized in this manner (43-45). Attenuated mutant Listeria monocytogenes containing foreign plasmid DNA were introduced into a macrophage cell line and showed expression of reporter proteins and subsequent antigen presentation (45). Similarly, attenuated Shigellaflexneriwhich is deficient in cell wall synthesis is also capable of DNA delivery (42,43). An E. colibased bacterial carrier also represents a promising delivery route, with advantages including the improved safety of utilizing non-pathogenic E. coli, the ability to easily manipulate its genome, and the simplicity of attaining high plasmid copy number (42,55). E. coli is, however, still subject to the limitations of host immunity. Common to all these bacterial vectors is high enough efficiency to mediate therapeutically significant delivery of genes, as well as weaknesses shared by viral vectors including pathogenicity, immunogenicity, and difficulty in targeting.

Liposome Formulations with Bacterial Mechanisms: the Next Generation Since risks and possible complications accompany the use of live bacteria, it is appealing to take only the necessary components from these intracellular invading organisms and design a non-bacterial delivery vehicle. The hemolysin of Listeria monocytogenes, listeriolysin Ο (LLO), is the well-characterized protein that is secreted by the intracellular parasite to escape from the endocytic compartment (4648). LLO is a soluble protein of 58 kD that is suggested to form a pore in



189 membranes. It belongs to the "thiol-aetivated, pore-forming hemolysin" family, which includes perfringolysin O (PFO), pneumolysin Ο (PLO), and streptolysin Ο (SLO). These hemolysins share significant sequence homologies to each other, and all bind only to cholesterol-containing membranes, oligomerize within the lipid bilayer to form pores, and lyse membranes. LLO is unique among the thiol-activated hemolysins in that it possesses pH-dependent hemolytic activity; LLO is activated by low pH as well as reduction of the unique cysteine (Cys 484) in the amino acid sequence (48,49). The Listeria gene for LLO, hly, has been cloned and was the first virulence gene identified in Listeria (50,51). A mutant, hly- Listeria cannot invade the cytosol of host cells; instead they die in the lysosomes, demonstrating that LLO is necessary for escape of Listeria into the cytosol (50,51). Portnoy and colleagues have demonstrated that when the hly gene is introduced into Bacillus subtilis, it confers the ability to escape from endocytic compartments (52), which is not inherently present in B. subtilis. This evidence suggests the hypothesis that a particulate carrier which releases LLO into the endosome may be able to disrupt endosomal membranes and deliver into the cytosol its internalized contents.

Early Work on a New Non-viral, Non-bacterial Liposome: Listeriosomes This strategy has been recently tested by designing a new liposomal delivery vehicle that contains LLO (53). This new cytosolic delivery system incorporates the endosomolytic activity of LLO into pH-sensitive liposomes which can destabilize and leak their contents along with LLO upon protonation of their pH-sensitive component. The lipid formulation used does not interact with LLO and retains the LLO molecules inside liposomes at neutral pH, such that release is dictated by acidification in the endosomes, where LLO is then activated to form pores in the endosomal membranes. LLO is co-encapsulated inside liposomes with macromolecular cargo to create a LLO-containing liposome, or listeriosome, and its ability to deliver macromolecules into the cytosol of macrophages has been investigated. Testing of listeriosomes to deliver molecules of molecular mass up to 50 kD has shown dramatic enhancement of the release of fluorescent dyes, proteins (53), and 20-mer oligonucleotides from endosomes into the cytosol (unpublished data). This new delivery system can be utilized as a general delivery vehicle for many membrane-impermeant macromolecular drugs provided they can be encapsulated inside liposomes with reasonable efficiency. The unique ability of listeriosomes to put antigenic proteins into the cytosol makes them optimal for targeting antigens into the MHC class I-mediated cytosolic pathway of antigen presentation and Τ cell activation for vaccination applications. Additionally, targeted listeriosomes have the potential to serve as carriers of various toxins for use in chemotherapy. The versatility of listeriosomes would allow for the incorporation of a variety of cargos, from proteins to genes, without compromising delivery characteristics. While LLO incorporation pioneered delivery using an isolated endosomolytic component of facultative intracellular bacteria, conjugation of PFO to DNA using a


190 biotin-streptavidin bridge has also been reported to yield enhanced levels of gene expression (54). However, the LLO protein has several beneficial inherent regulatory mechanisms including optimal activity at the pH of endosomes (48,49), which make it an ideal endosomolytic agent. Furthermore, since Listeria has evolved to be intracellular while Streptococcus pyogenes or Clostridium perfringens extracellular bacteria, LLO should be better suited for endosome disruption for efficient and safe cytosolic delivery than SLO or PFO.


Conclusion The ability to deliver a variety of drugs and macromolecules into the cytosol is essential to many areas of basic biology and pharmaceutics. Potential applications include polypeptide delivery of antigenic proteins to induce cell-mediated immunity, chemotherapy using polypeptide- or recombinant protein-based drugs, delivery of oligonucleotide inhibitors in cell biology and in clinical settings, and plasmid DNA delivery in gene therapy. Liposomes in recent decades have gone through several transformations and will need significant further investigation to be used as a general delivery system for a variety of macromolecular therapeutic agents. In addition, modifications and customization for each application, depending on particular drugs and target cells, will be still necessary. Among the promising new strategies, the recent approach using the LLO-based mechanism presents an exciting solution to the limitations of liposomal cytosolic delivery. Several key questions, including (i) if the LLO-base delivery vehicles perform in vivo as well as in tissue culture and (ii) if nucleic acid-based macromolecules of various sizes can be delivered as efficiently as the proteinacious compounds, must be addressed in order to establish intracellular bacterial-based mechanisms of delivery as a new paradigm in the cytosolic delivery of macromolecules.

Acknowledgements The authors would like to thank the University of Michigan and the College of Pharmacy for financial support (Rackham Faculty Research Award and Vahlteich Research Fund). Gretchen Larson is a Regent's Fellow at the University of Michigan and a F. Lyon's Fellow at the College of Pharmacy. This work has been also supported by the grants to K.-D. Lee from NIH (AI42084 and AI42657).

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Modified Liposome Formulations for Cytosolic Delivery of

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