Clearance properties of liposomes involving conjugated proteins for targeting

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Abstract

This review examines methods of protein conjugation onto liposomes and the effects of surface bound protein on the liposomes' biological behavior. It is evident that the presence of a conjugated protein significantly alters the attributes of targeted liposomes. Specifically, protein conjugation can result in dramatic increases in liposome size, enhanced immunogenicity, and increased plasma elimination. Techniques are discussed for preventing some of the physical (size) and biological (immunogenic) alterations involving the use of PEG-lipids and drug loaded liposomes. In addition, the advantages of conjugating antibodies via carbohydrate moieties, to minimize changes in antibody binding and tertiary structure as well as effectively decreasing plasma elimination, are also discussed. It is, however, apparent that the accessibility of targeted liposomes to extravascular sites is a key step that will require further study and it is, therefore, anticipated that with the development of novel ligands and novel ligand–liposome interactions, the therapeutic utility of targeting strategies will likely be realized.

Introduction

Treating human disease with targeted drugs will have major impacts on increasing disease-free survival and improving patient quality of life. The treatment of illnesses such as cancer with targeted drugs, however, has been an elusive goal. The targeting of drugs using liposomes has been hampered by several biological processes, discussed in Section 1.4, which have played critical roles in the limited success of targeting. As a result, we are faced with difficult challenges to develop practical solutions to overcome these obstacles. Future success of targeted liposomes will require further characterization of the physical and chemical attributes of ligand–liposome complexes including a better understanding of how these attributes dictate biological behavior in general and target site delivery and binding in particular. This section will discuss what we believe are the goals and therapeutic objectives for developing target liposomes.

The development of successful targeting agents will have dramatic impacts for a number of medical applications, particularly the diagnostic capabilities of imaging agents [1]and improving the specificity of highly toxic drugs used to treat neoplastic diseases [2]. We believe that by far the greatest benefits will occur with the use of chemotherapeutics to treat cancer. Currently, general protocols for the treatment of cancer rely on administering chemotherapeutic agents as either single agents or in combination with other drugs. The effective doses for these agents are, in most instances, administered at the drug's maximum tolerated dose. The unfortunate impact of treating at such high doses is side effects which can be life-threatening. The combination of drug toxicity and poor therapeutic activity at nontoxic doses means that most chemotherapeutic agents exhibit very narrow therapeutic windows. However, having the ability to localize drug to a specific target site should provide a major advancement for anti-cancer drug therapy, as it will allow for more effective treatments to be given at doses that are better tolerated.

The success of nontargeted liposomes for localizing drug to target sites is apparent from the number of formulations in advanced clinical trials and the fact that they are used to treat a number of diseases (Table 1). In particular, market approval for two liposomal formulations, Doxil™ and DaunoXome™, has been achieved for the treatment of AIDS related Kaposi sarcoma. The main characteristics which have contributed to this success are a consequence of the liposomes' ability to decrease drug toxicities, to increase drug circulation lifetimes, and to inadvertently accumulate in sites of disease and infection 3, 4, 5, 6. We believe the latter is a key reason why liposomes are an excellent choice for targeted delivery vehicles. Studies from our laboratory and others, however, suggest that the incorporation of ligands onto liposomes for targeting purposes reduces the natural ability of the liposomes to access target sites 7, 8, 9.

The pharmacological research goal of developing targeted liposomes is to increase target site accessibility and, ultimately, target diseased cells outside of the vasculature. In this context targeted liposomes will require access to the target and an ability to interact with and bind target cells. Binding will be dependent upon the presence of a ligand which has a high affinity or specificity for a marker (receptor) on the surface of target cells. The most accessible target cell populations reside in the vasculature compartment and include lymphocytes and erythrocytes 10, 11. Although therapeutically important goals can be defined for targeting cells within the vascular compartment, the focus of this review will be on the more challenging goal of targeting to extravascular sites. Success in achieving extravascular targeted drug delivery has been hampered for reasons which include liposome circulation lifetimes, accessibility to the target site [7]and limitations in the manufacturing procedures used to prepare antibody-conjugated liposomes. Efforts directed to overcome these hurdles will be summarized in this review, which focuses on the use of antibody ligands and liposomal delivery systems for the treatment of solid tumors. We will also speculate on the development of strategies to improve or control the pharmacodynamic behavior of the targeted liposome such that the attributes required for proper targeted liposome function are maintained. The consequences regarding repetitive dosing and the immunogenicity of such liposome formulations will also be considered.

For reasons that are difficult to define, nontargeted liposomal systems have been reasonably effective at accessing target sites (referred to as passive accumulation). In particular, liposomes with long circulation lifetimes are known to accumulate in extravascular disease sites including sites of infection, inflammation, and tumors 3, 4, 5, 6. Improvements in drug localization are achieved, therefore, in the absence of any targeting information as a consequence of passive delivery of drug loaded liposomes (Fig. 1D). Liposome accumulation can result in three to 100-times more drug delivery to a target site compared to the injection of the same dose of free drug. However, the majority of localized nontargeted liposomes do not interact with target cells directly [12]. It is suggested that therapeutic activity is a consequence of drug release from liposomes within the disease site, a process that does not require direct binding or association with diseased cells (Fig. 1E).

The facilitation of the binding of the drug carrier or liposome to target cells through the use of ligands to increase localization of drug and target cell killing is referred to as active targeting (Fig. 1G). Specificity of actively targeted liposomes is dependent upon the ligand's affinity for a target cell marker. As with passive targeting it is anticipated that long circulation lifetimes will be required in order to maintain target site accumulation of these carriers (i.e. passive targeting capabilities), an essential requirement for target cell access.

One method of incorporating target specificity in a liposome is accomplished by the use of a nonspecific intermediate, or bridging protein, to associate the drug loaded liposomes with target cells. This intermediate has specificity for the target cells and an affinity for a component on the liposome. This approach is best exemplified by antibodies, modified with biotin and liposomes which have covalently attached avidin (or streptavidin), a biotin binding protein (Fig. 2A). The use of an intermediate protein for targeting liposomes has been referred to as a two-step approach, as it requires the administration of the intermediate protein and liposome separately. Customarily, the intermediate protein is administered first to allow accumulation and binding to the target cells. At a pre-determined time, when the maximum target cell to blood ratio is achieved, the liposomes are administered. The liposomes can then bind the intermediate protein labeled target cells. Several advantages of two-step targeting approaches include (i) the use of simple lipids and/or well defined liposomes to achieve targeting, (ii) no need for coupling procedures to be optimized for each targeting protein used, (iii) characterization of binding/labeling of target cells in the absence of liposomes, and (iv) the lack of altered liposome biodistribution resulting from the attachment of large targeting ligands. Two-step approaches, however, are not necessarily viable options because of difficulties in developing clinical studies where two active components must be independently characterized pharmacokinetically. Further, the advantage of using small ligands on liposomes was thought to result in reduced immunogenicity. Recent evidence, however, indicates that even small ligands (such as biotin) can be immunogenic [13]. Finally, the targeting intermediate used can only be directed against cell surface markers that are recycled at a slow rate, either through shedding or internalization. Therefore, it is likely that binding will occur in the absence of efficient intracellular delivery.

The second approach to achieve specificity is by preparing liposomes with targeting ligands directly conjugated onto the membrane surface (Fig. 2B). Several types of ligands can be incorporated such as peptides, glycoproteins, or vitamins. The latter are used mainly for their smaller size and ease in which they can be attached to liposomes [14]. The main focus of such research has, however, been on the use of antibodies. Antibodies are commonly used as they are inexpensive, easy to produce in large quantities, well characterized for pre-clinical and clinical use, have a high affinity for their antigens, and are available against a wide variety of unique, over-expressed, antigenic determinants located on cells. While many different ligands, other than antibodies, can be conjugated to a liposome, coupling techniques rely on a limited number of covalent or noncovalent reactions. Covalent strategies use a variety of bifunctional cross-linkers and typically involve modification of the ligand and subsequent conjugation to preformed liposomes containing a modified and reactive phosphatidylethanolamine (PE). Noncovalent strategies involve ligand binding mediated through biotin–streptavidin or similar interactions involving proteins that can bind the Fc domain of IgGs, such as protein G or protein A. Other approaches have used detergent dialysis to incorporate antibodies that have been modified with hydrophobic groups such as fatty acyl chains [15]. The latter approach has since been simplified by the development of lipid tagged single chain antibodies. This approach is advantageous as it avoids the chemical reactions typically required when attaching antibodies to preformed liposomes [16].

Considering the variety of approaches that have been developed to achieve target cell-specific delivery of liposomes, it is curious as to why over 20 years worth of scientific research has not led to the development of a single therapeutically and clinically viable targeted liposomal drug. The answer is simple in that the technology is not sophisticated enough. In the absence of improved technology, the methods used to prepare targeted liposomes are, however, appropriate for establishing proof-of-concept. To date such proof-of-concept data has been limited to only a few studies demonstrating improved therapy for targeted formulations directed against diseases in the lung 17, 18. We believe that there are two significant concerns associated with the use of liposomes for targeting cells in extravascular sites. First, the targeted liposomes are often rapidly eliminated from the circulation. Rapid plasma elimination decreases passive accumulation of targeted liposomes in the diseased tissue, resulting in low target site access and low target cell contact [7]. It is anticipated that if we can successfully increase plasma levels then increases in accumulation within target sites will result. The second concern relates to the potential of targeted liposomes to interact with the target cells after entering an extravascular site. In order for this to be achieved one must (i) ensure stable targeting ligand/liposome interactions and (ii) demonstrate binding to the target cell. In order to develop methods/approaches to target liposomes, a review of the methods presently used to attach targeting ligands to liposomes is useful. This information is particularly helpful when considering the biological factors that can reduce/block the potential for these systems to be used systemically. Although the previous sections provide a general description of targeting methods, it is necessary to provide details about commonly employed linker methods prior to assessing how coupling conditions affect liposome structure and biological behavior. In general, these coupling procedures add moieties that elicit immune reactions, engender liposome–liposome crosslinking and decrease affinity of the targeting ligand for its receptor. In turn, these effects result in more rapid liposome clearance, poor target site accessibility, and poor target site binding.

Section snippets

Methods of target ligand incorporation into liposomes

As mentioned previously, two methods predominate for attaching ligands onto a liposomal surface, (i) covalent conjugation, which is the direct coupling of a ligand onto the liposome and (ii) noncovalent conjugation, which relies on the interactions of an intermediate to bind the targeting ligand to the liposome.

Biological interactions: Effect of target ligand attachment on liposome behavior in vivo

It is now well established that modification or conjugation of protein on the liposome surface can have significant effects on the acute biological behavior of the liposome following systemic administration. Also, the presence of attached protein and reactive groups will lead to an immune response. The effects of greatest interest are those that result in faster liposome elimination. To overcome increased plasma elimination the next section considers some established methods that have been used

Conclusion

Regardless of the targeting ligand used, there are at least two fundamental steps that must be achieved for targeted liposomal systems to become therapeutically useful. First, targeted liposomes must have reasonable plasma circulation times to allow access to a tumor site. Ample evidence demonstrates that liposomes accumulate in disease sites and in sites of infection and inflammation 3, 4, 5, 6, an effect primarily attributed to the presence of leaky vasculature in regions of necroses and

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      We speculate that the higher clearance rate of RGDfK-PLDs could be attributed to the higher exposure of RGDfK on liposome surface during circulation. Given the role of opsonization in recognition and uptake of liposomes (Moghimi and Patel, 1989) and in accordance with Sancey et al. (2007) results, RGDfK-PLD could widely be recognized by opsonins and finally opsonized by fixed and free RES cells (Tardi et al., 1998) or easily be recognized by integrins expressing cells residing in non-tumoral tissues, most importantly liver and spleen, whereas limited exposure of RGDyC or RGDf[N-Met]K could reduce both opsonization and direct recognition of RGDyC-PLD and RGDf[N-Met]K-PLD by RES cells and subsequently displayed lower clearance rate compared to RGDfK-PLD. Interestingly, although RGDyC-PLD indicated same plasma pattern as what was observed for RGDf[N-Met]K-PLD, it failed to reach the same tumor levels (Fig. 5B) and also caused more side effects (Fig. 4C).

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