Topology of Surface Ligands on Liposomes: Characterization Based on the Terms, Incorporation Ratio, Surface Anchor Density, and Reaction Yield

Abstract

The surface topology of ligands on liposomes is an important factor in active targeting in drug delivery systems. Accurately evaluating the density of anchors and bioactive functional ligands on a liposomal surface is critical for ensuring the efficient delivery of liposomes. For evaluating surface ligand density, it is necessary to clarify that on the ligand-modified liposomal surfaces, some anchors are attached to ligands but some are not. To distinguish between these situations, a key parameter, surface anchor density, was introduced to specify amount of total anchors on the liposomal surface. Second, the parameter reaction yield was introduced to identify the amount of ligand-attached anchors among total anchors, since the conjugation efficiency is not always the same nor 100%. Combining these independent parameters, we derived: incorporation ratio surface anchor density reaction yield. The term incorporation ratio defines the surface ligand density. Since the surface anchor density represents the density of polyethylene glycol (PEG) on the surfaces in most cases, it also determines liposomal function. It is possible to accurately characterize various PEG and ligand densities and to define the surface topologies. In conclusion, this quantitative methodology can standardize the liposome preparation process and qualify the modified liposomal surfaces.Key words active targeting; surface ligand density; incorporation ratio; surface anchor density; reaction yield; surface topology Nanotechnology has the capability to deliver drugs to specific cell types, but it is important to maximize therapeutic effects and minimize unexpected adverse effects, for this technology to be effectively used.1-4) In using nanoparticles for specific drug delivery, they need to have both a stealth function to prevent non-specific recognition by the reticuloendothelial system (RES), 5,6) and an active targeting function to allow them to bind to the specific cell type of interest. 7-9)Polyethylene glycol (PEG) modification is frequently used to confer a stealth function to nanoparticles. 5,6,10) For active targeting, specific ligands for bioactive molecules such as nucleic acids, peptides, proteins, and antibodies are attached to the surface of nanoparticles via a PEG spacer.11) Many attempts have been made to develop various types of specific ligands for use in active targeting, which is critical for targeting non-fenestrated tissues such as the brain, lung and related tissues. [12][13][14] The procedures used to prepare ligands that are used in modifying nanoparticles are relatively complicated because this increases the number of steps in the preparation process that can lead to conditions where ligand molecules can be unstable.15) Therefore, optimal reaction (modification) conditions and methods for quantifying the density of the attached ligand that is attached to the nanoparticles need to be evaluated precisely.Problems are usually encountered in evaluating both the density of the liposomal ligands and the PEG, since a variety of m...

Targeted brain delivery of methotrexate by glutathione PEGylated liposomes: How can the formulation make a difference?

“…The discrepancy in the extent of in vivo MTX release can likely be attributed to different PEGylation methods used in the two studies. Previously, 5% PEG was incorporated into both HSPC and EYPC based PEG liposomes using the pre-insertion method, which led to about half of the PEG being oriented outward from the membrane and the rest toward the inside of the liposomes [23]. In the current study, 4% PEG was post-inserted, leading to PEG being present mainly on the outer side of the liposomes, with around 4.5% PEG presented on the surface and 0.5% PEG facing inwards, including the 1% PEG pre-inserted.…”

Size-Dependency of the Surface Ligand Density of Liposomes Prepared by Post-insertion

“…In our methodology, the term 'incorporation ratio' is introduced as a parameter that reflects the accurate surface ligand density. 33) In Chart 1, to analyze the incorporation ratio of the inserted (functionalized) liposomes a post-insertion procedure was employed using plain liposomes with a 5-FAM-PEG 2000 -DSPE conjugate lipid (5-FAM conj. ), in which the liposomal surface was modified with a fluorescently labeled functionalized conjugate.…”

“…The calculation was based on the assumption that the conjugates are exposed outward/inward from the membrane with a theoretical ratio 55/45 on the pre-inserted liposomes, i.e., incorporation ratio=(incorporated 5-FAM conj.×(55/100))/(lipids mixture+incorporated 5-FAM conj.). 33) Surprisingly, small differences in the incorporation ratios in eight different sizes of fractional liposomes suggested the absence of any size-dependency trend during the formation of pre-inserted liposomes. This result may be caused by homogeneous lipid constituents in the initial hydrated liposomes that form the liposomes during the sonication process.…”

“…36) The parameter, 'incorporation ratio,' is introduced to define this density. 33) In an analysis of these ratios, our new discovery shows that when liposome size decreases, the incorporation ratio is not constant but rapidly increases. Since liposomes with smaller sizes promise to become more important in the near future, a significant underestimation of the incorporation ratio may be misleading and lead to erroneous conclusions regarding their bioactivities.…”

“…[17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] To address this issue, a method for estimating and calculating these values is needed and the liposomal preparation should be standardized, since these factors affect the density of the ligand on the surface of a particle. 33) Considering the clinical aspects of regulatory science (medicine), the characterization of liposomal surfaces guarantees the quality of a medical application. An accurate value for the surface ligand density provides an accurate assessment of the degree of binding affinity for a modified liposome.…”