Thursday, May 31, 2012

Mannosylated Liposomes

We are continuing to review publications related to mannosylated liposomes. This discussion will be updated with any pertinent information as we find it or whenever new information is published.

Summary
Mannose receptor binding is one of the oldest proposed mechanisms for liposome targeting through surface modification of the liposomes. The increasing number of both cell-surface-associated and soluble mannose-binding proteins that have since been discovered reveal the complexity of the mechanism(s) involved in enhanced uptake of mannosylated liposomes [1]. Yet, several publications present convincing data on the ability of liposomes with various different conformations of surface bound mannose molecules to target mannose-receptor positive cells both in vitro and in vivo. Unfortunately, most of these proven formulations have not been applied to selective macrophage depletion by encapsulation of clodronate in these liposomes. The clodronate liposome field, in particular, has been plagued by the introduction of a liposome formulation prepared in the presence of p-aminophenyl mannose, but without covalently coupling this modified sugar to the liposome surface. The profusely cited reference to this liposome preparation claimed to target mannose-receptor positive cells in the brain as well as deliver liposome cargo across the blood-brain barrier. We find the data in this paper to be very weak and cannot confirm that the formulation was ever appropriately characterized with respect to the presence of mannose capable of targeting the liposome to mannose-binding proteins. While some stronger data suggesting that this formulation may target mannose-receptor positive cells has been published, others find no evidence of targeting by this formulation. We conclude that studies specifically designed to characterize and assess the targeting ability of this formulation are required to justify the use of this formulation in targeting mannose-receptor positive phagocytes. Until these studies are completed and published, we recommend that formulations containing covalently-anchored mannose and demonstrating the ability to selectively bind mannose receptors be utilized when experimental design calls for mannose receptor targeting.
Many different mannosylated lipids have been synthesized which effectively display mannose residues on the surface of liposomes suitable for binding to mannose receptors as judged by Con A agglutination and competitive inhibition of mannose receptor binding by mannose or mannose-containing macromolecules. These demonstrations, however, are more difficult to interpret in vivobecause of the multiple mechanisms by which phagocyte uptake of mannosylated liposomes is enhanced. The structures of these mannosylated lipids are shown on this page along with a brief discussion of the published characterization and behavior of liposomes containing these derivatives. While the rationale for the design, synthesis and biophysical properties of these molecules may not seem directly applicable to clodronate liposome studies, there is a great deal of useful information in these papers on the rational design of surface-modified liposomes which will be invaluable when interpreting mannosylated clodronate liposome depletion data as well as when attempting to deplete specific mannose-receptor-positive phagocyte subsets.
Despite the large number of publications by those attempting to deplete mannose-receptor-positive phagocyte populations with liposomal clodronate prepared in the presence of p-aminophenyl mannose, combined with as many, or more, papers from those designing mannosylated liposomes for targeting cells displaying macrophage receptors, thus far, we have found only one paper describing mannosylated clodronate liposomes that were confirmed to target mannose receptors in vitro. Therefore, there is a great deal of work to be done with regard to evaluating the use and advantages of mannosylated clodronate liposomes.
We are awaiting publication of data from one group to whom we supplied covalently-coupled mannosylated liposomes; they have indicated achieving successful targeting and depletion of a mannose-receptor positive phagocyte subpopulation. By compiling the information on this page, it is our intention to equip everyone with the basic tools necessary for successful, unequivocal mannose receptor targeting using mannosylated clodronate liposomes. 
Introduction. Multiple studies have shown that modifying the surface of liposomes with mannose [1] increases the uptake of these liposomes in phagocytes bearing a mannose receptor mechanism [2, 3, 4, 15] as far back as the mid-1970′s [8]. Obviously, this is not a new idea although Juliano’s first efforts actually produced a liposome with the lectin (carbohydrate-binding protein) on the surface rather than the sugar. While we are focusing on mannosylation, many other publications describe liposome targeting using other carbohydrates. The significant amount of data on mannose and other carbohydrate targeting should not be confused with being able to selectively target macrophage subpopulations which may express mannose receptors. Mannosylated-liposome uptake is enhanced by other mechanisms unrelated to mannose receptor binding, such as recognition by mannose-binding protein (MBP or MBL) , which activates complement and initiates uptake by complement receptor mediated phagocytosis [5, 11]. While all liposomes activate complement and bind other serum opsonins to some extent, certain types of liposomes (i.e. 100 nm DSPC/cholesterol liposomes) bind much less complement than others, thus exhibit extended circulation times post-i.v. injection in vivo.However, when mannose or other carbohydrates were covalently attached to the liposome surfaces, they are rapidly cleared from the bloodstream [6]. This has even been shown for so-called “stealth” liposomes, known for their ability to avoid immediate phagocyte uptake [71].
Why Mannose? Mannose is one of the carbohydrate components of many bacterial and viral cell surfaces, therefore the ever-efficient, highly redundant immune system has evolved multiple mechanisms for identifying pathogens based on mannose recognition. The animal and plant kingdoms likewise utilize carbohydrate recognition signalling mechanisms including mannose residues. Many publications evaluate other carbohydrates as targeting mechanisms for various cell types, however mannose targeting to phagocytes appears to be one of the more specific mechanisms identified to date [6]. Mammalian cell surface identification molecules based on mannose binding, such as the ICAM family of leukocyte adhesion molecules, target the SIGN family of mannose receptors to accomplish self-recognition in vivo [12, 13, 14]. Several other mannose-binding molecules have been identified, but not all of them have been connected to their biologically relevant ligands and functions [3]. There are also a large bodies of work focusing on using mannosylated liposomes as an adjuvant, for non-specific immunostimulation and for nucleic acid delivery, the mechanisms all of which are somewhat outside the focus of this discussion [15, 16, 17, 18, 19]. However, these applications have provided a multitude of methods for synthesizing mannosylated-lipid conjugates that can be included into mannosylated-liposomal clodronate formulations.
Mannose- and Mannose-Liposome-Binding Serum Components. The abundance of both cell associated and soluble mannose receptors in serum contributes to the lack of specificity of mannose targeting [9]. As mentioned earlier, mannose-binding protein, present in mammalian serum, is very effective at complement activation when MBP is bound to mannose. Several studies describe the soluble mannose receptor (sMR)which is the extracellular portion of the  macrophage mannose receptor (MR) released by macrophages for transferring macrophage-processed antigens to antigen-presenting cells (APC) [20]. While MBP requires the association of several subunits, each possessing a single carbohydrate binding domain (CRD) for high affinity binding, sMR possesses 8 CRDs on a single molecule. Therefore sMR has no requirement for self-association in serum before binding occurs as does MBP.
MBP is closely related to surfactant proteins A and D (SP-A, SP-D) which also bind mannose in the lung. Therefore, intrapulmonary administration of mannosylated liposomes will also result in binding of mannose-specific proteins that will compete with MR+ alveolar macrophages and other MR+ pulmonary phagocytes for the mannose on the liposome surface [22]. The receptors and functions of SP-A and SP-D have been the subject of conflicting reports and are still not clearly understood [91], furthermore these soluble mannose receptors are found in several locations outside the lung [92] making it somewhat more difficult to take into account how these lectins will affect mannosylated liposomes.
Complement component, C3 , has been shown to covalently bind mannose during its activated state, although this has not been demonstrated in vivo [21]. Antibodies bind both specifically (anti-phospholipid antibodies) and non-specifically to liposomes in vivo [7]. While not directly addressed in any of these publications, it seems that antibodies generated from previous exposure to various mannose-displaying pathogens may recognize some mannosylated liposomes due to their similar surface properties. We highlight these soluble mannose receptors, because when designing a targeted liposome, as with any other liposome for use in vivo, the first consideration is how the liposome will interact with the biological milieu before it reaches its potential target cell.
In summary, the propensity for serum components binding to liposomes is enhanced by the presence of both mannose-specific and non-specific components when the liposomes are surface-modified by mannose. Most of this enhanced binding correlates to enhanced uptake of liposomes in the liver since removing foreign particles from the bloodstream is one of the liver’s primary functions. In fact, Ghosh, et. al. and other groups have demonstrated that mannosylated liposomes are preferentially phagocytosed by non-parenchymal liver cells, including Kupffer cells, while galactosylated liposomes tend to be associated with hepatocytes [41].
Cell-Surface Mannose Receptors. When developing a mannose-targeting rationale, the macrophage mannose receptor (formerly MMR, now MR or CD206) is usually the targeting goal. This receptor was originally discovered and characterized from macrophages, but this receptor has since been found on dendritic cells, certain endothelial cells and as a non-cell-bound soluble form which has been shown to migrate from macrophages to antigen-presenting cells (APC) as discussed above [43, 44]. A structurally and functionally similar receptor, Endo-180, was first identified on fibroblasts, but has now been identified on macrophages and endothelial cells in all tissues. Endo-180 appears to function in regulation of extracellular matrix catabolism and structuring. DEC-205, a mannose receptor first identified in maturing dendritic cells and one of the receptors which binds the soluble from of phospholipase A2, M-PLA2R, also specifically binds mannose-containing molecules.  This family of cell surface mannose receptors all operate by the clathrin-mediated (coated-pit) endocytic mechanism which would limit the size of the liposome that could be internalized to a few hundred microns. However, MR itself is unique in that it also initiates actin-mediated phagocytosis although some speculate that MR requires cooperation with other receptors to accomplish phagocytosis. DEC-205 and M-PLA2R are not likely to play a significant role in mannosylated liposome targeting, but their ability to bind specific mannose-containing molecules may recruit mannosylated liposomes to the cell surface increasing the likelihood that the liposome will come into contact with the phagocytic mannose-receptors.
In addition to MR and Fc (antibody) receptors which allow dendritic cells to phagocytose pathogens, a distinctly different receptor which recognized mannose was first identified on these cells. This receptor was found to mediate the interaction between dendritic cells and T cells which allows the transfer of antigenic peptides produced by dendritic cells (from ingested pathogens) to the T cells (for antibody production) through the membrane-associated MHC shuttle. The receptor ligand on T cells was identified as ICAM-3 and the receptor was designated DC-SIGN as it had only been identified on dendritic cells [13, 14]. Of course, related receptors (SIGNRs) were later identified in many tissues including splenic red pulp and peritoneal macrophages (SIGNR1). Mannosylated-liposome targeting to this receptor demonstrates the requirement for complement receptor (CR3) participation in the phagocytosis of the liposomes suggesting that SIGN receptors do require cooperative support from other phagocytic receptors as has been speculated for MR [12]. Langerin is another SIGN-like mannose-binding receptor speculated to be a target for mannosylated liposomes.
Clearly, a variety of potential mannose-targeting sites exist on the surface of many cell types including those susceptible to depletion by liposomal clodronate. Although all of these sites may not initiate phagocytosis of clodronate liposomes, mannosylated clodronate liposomes may bind to any of these sites ensuring that the liposomes are in close proximity to cell-surface phagocytic receptors thus increasing the probability of liposome phagocytosis.
Is there a Rationale for using Mannosylated Clodronate Liposomes? Given that mannosylated liposomes are expected to demonstrate enhanced uptake by all (not only those with mannose receptors) accessible phagocytic cells due to their increased level of opsonization, it is difficult to predict if the pattern of macrophage depletion will be substantially different in vivo. Buiting, et. al. report no significant differences in the biodistribution of clodronate liposomes of differing lipid compositions including the p-aminophenyl mannose formulation as well as a colvalently linked mannosyl-DOPE conjugate [48]. With regard to the covalently attached mannose liposomes, DOPE is a non-bilayer forming lipid which is known to be disruptive to liposomes under many conditions and is suspected to have demonstrated similar disruptive behavior as described for Weissig, et. al. below. Successful targeting with the PE-linked mannose studies employ DPPE or other bilayer-preferring PE species. Additionally, this mannosyl-DOPE conjugate does not include a flexible spacer which extends the mannose away from the liposome surface, the importance of which is also discussed below. Furthermore, most mannosylated-lipid studies report that two or more mannose residues per lipid are required for optimal mannose-receptor binding while this conjugate has a single mannose molecule per lipid. Taken all together, we believe that this covalently-linked mannose liposome formulation was not optimized for successful targeting. Yet, many mannose-conjugated lipid derivatives (shown below) do demonstrate selective uptake in vitro and different biodistribution patterns in vivo, and there is a study showing that mannosylated (ManDog) clodronate liposomes are effective for depleting immature dentritic cells in vitro, while non-manosylated liposomes have no effect as discussed in detail below. These data suggest that depleting atypical subsets of phagocytes with well-characterized mannosylated clodronate liposomes is an intriguing possibility worth investigating.

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