Relationships between Liposome Properties, Cell Membrane Binding, Intracellular Processing, and Intracellular Bioavailability

Abstract

Positive surface charge enhances liposome uptake into cells. Pegylation, used to confer stealth properties to enable in vivo applications of cationic liposomes, compromises internalization. The goal of this study was to determine the quantitative relationships between these two liposome properties (separately and jointly), liposomes binding to cell membrane, and the subsequent internalization and residence in intracellular space (referred to as intracellular bioavailability). The results, obtained in pancreatic Hs-766T cancer cells, revealed nonlinear and inter-dependent relationships, as well as substantial qualitative and quantitative differences. The proportionality constant K of intracellular and membrane-bound liposomes at equilibrium (i.e., I(eq) and B(eq)) showed a positive triphasic relationship with surface charge and a negative biphasic relationship with pegylation. Near-neutral liposomes showed little internalization of the membrane-bound moiety, increasing to a constant K value for medium charge liposomes (+15 to +35 mV zeta potential), followed by a further increase for highly charged liposomes (greater than or equal to +46 mV). The decline of pegylation with K value showed a breakpoint at 2%. The negative consequences of pegylation (%PEG) were partially offset by increasing charge (ZP). The best-fitting regression equations are: B(eq) = -1.36 × %PEG + 0.33 × ZP; I(eq) = -1.52 × %PEG + 0.34 × ZP. It suggested that 1% pegylation increase can be offset with 4 mV ZP. The differences are such that it may be possible to balance these parameters to simultaneously maximize the stealth property and intracellular bioavailability of cationic liposomes.

Predictive Models of Diffusive Nanoparticle Transport in 3-Dimensional Tumor Cell Spheroids

“…The second method measures the total amount of cell-associated NP after incubation at 4°C which reflects the cell surface-bound NP, and the amount of cellassociated NP after incubation at 37°C which reflects the sum of surface-bound and internalized NP (21)(22)(23); the difference between these two values represents the internalized NP. We previously confirmed the 4°C measurement reflects the surface-bound positively charged liposomes (6). The effectiveness of the 10×-trypsin stripping method was confirmed using confocal microscopy to compare the subcellular NP distribution before and after the treatment.…”

“…The sizes of the three NP used in the current study covers the range of nano-size therapeutics, e.g., macromolecule drugs such as antibodies and proteins (5-20 nm) and nanoparticle drug carriers (e.g., 85 nm for Doxil and 130 nm for Abraxane; 1, [40][41][42]. Surface charges of the three NP are also within the range of nano-therapeutics −12 mV for Doxil, −30 mV for Abraxane, >40 mV for cationic liposomes (6,(42)(43)(44). The different NP properties (charge, size, materials) led to differences in NP-cell interactions, as shown by their different k on (>3-fold), k in (>8-fold), and B max,single cell (>80-fold) values.…”

“…Fusogenic lipids are used to fuse with the endosomal membrane to facilitate endosomal escape (5). We recently showed that surface charge and pegylation have opposing quantitative effects on NP binding to cell surface and internalization such that the effect of one parameter is offset by the other (6).…”

Liposomes containing bile salts as novel ocular delivery systems for tacrolimus (FK506): in vitro characterization and improved corneal permeation

“…29 Cellular uptake was correlated with surface charge and increased with increasing positive charge. 30 Therefore, the negatively charged nanoparticles adsorbed poorly onto the cell membrane, resulting in reduced SDHCEC uptake of liposomes containing bile salts compared with those containing cholesterol. However, cellular uptake of liposomes containing sodium glycocholate and those containing cholesterol was similar.…”

Interactions of antitumour Sialyl Lewis X liposomes with vascular endothelial cells

“…400 μL of short time-treated cells were collected and solubilised with 20 μL of 10% Triton X-100 and sonicated for 30 min, resulting in total cell-associated liposomes. Long time-treated cells were centrifuged at 1000 g (4°C, 20 min) to separate precipitated internalised fractions of liposomes and membrane-bound fractions in the supernatant [35]. After solubilisation with Triton X-100, the fluorescence of BODIPY-PC in the resulting solution was measured in a quartz cuvette under constant stirring using Hitachi F4000 spectrofluorimeter with excitation and emission band-pass 5 nm, λ ex 490 nm and λ em 506 nm.…”