Selective Endocytic Uptake of Targeted Liposomes Occurs within a Narrow Range of Liposome Diameters

Abstract

Cell surface receptors facilitate signaling and nutrient uptake. These processes are dynamic, requiring receptors to be actively recycled by endocytosis. Due to their differential expression in disease states, receptors are often the target of drug-carrier particles, which are adorned with ligands that bind specifically to receptors. These targeted particles are taken into the cell by multiple routes of internalization, where the best-characterized pathway is clathrin-mediated endocytosis. Most studies of particle uptake have utilized bulk assays rather than observing individual endocytic events. As a result, the detailed mechanisms of particle uptake remain obscure. To address this gap, we employed a live-cell imaging approach to study the uptake of individual liposomes as they interact with clathrin-coated structures. By tracking individual internalization events, we find that the size of liposomes rather than the density of the ligands on their surfaces primarily determines their probability of uptake. Interestingly, targeting has the greatest impact on endocytosis of liposomes of intermediate diameters, with the smallest and largest liposomes being internalized or excluded, respectively, regardless of whether they are targeted. These findings, which highlight a previously unexplored limitation of targeted delivery, can be used to design more effective drug carriers.

Keywords: TIRF microscopy; clathrin-mediated endocytosis; drug-carrier; internalization; liposome; targeted delivery.

Figure 1.. Targeting promotes penetration of liposomes beneath adherent cells.

A. The chimeric receptor, TfR-mEGFP-mSA, which was designed to recruit biotinylated liposomes to endocytic sites. B. Schematic of TIRF microscopy to examine internalization of liposomes by endocytosis from the adherent surfaces of cells. C-E. TIRF microscopy images of AP2 (JF646), chimeric receptor (EGFP), and liposomes (Texas Red DHPE) at the adherent surfaces of SUM159 cells. All channels have been contrasted equally across all three conditions. F. Average fluorescence intensity of the chimeric receptor on the plasma membrane. No significant differences were seen between groups of cells exposed to each population of liposomes. (t-test 0% vs 10%; p = 0.514; n = 107, 182) t-test 0% vs 20%; p = 0.353; n = 107; 166; t-test 10% vs 20%; p = 0.800; n = 182, 166). G. Total fluorescence intensity per area of liposomes present beneath cells. (t-test 0% vs 10%; p < 1x10⁻⁴; n = 107, 182) t-test 0% vs 20%; p < 1x10⁻⁴; n = 107; 166; t-test 10% vs 20%; p = 3.55x10⁻⁴; n = 182, 166). For F and G, three independent trials were acquired for each condition with a minimum of 15 cells imaged per trial. Error bars represent the standard error of the mean, where N is the number of cellular crops analyzed across all trials. Significance between conditions was identified using a two-tailed student’s t-test and a one-factor ANOVA with α = 0.05.

Figure 2.. Liposomes associate with clathrin-coated structures that have longer lifetimes.

A. A TIRF microscopy image at the plasma membrane of a SUM159 cell, gene edited to express a HaloTag on the σ-subunit of AP2, incubated with 1μM of liposomes (total lipid), which contained 10 mol% of biotinylated lipids. The dashed line represents the outer edge of the cell being analyzed. B. Fluorescence intensity as a function of time for an individual liposome, which colocalized with an individual clathrin-coated structure. The liposome and clathrin-coated structure decay in intensity over the same period of time, suggesting simultaneous departure from the TIRF field, as expected for internalization of a liposome by endocytosis. C. Montage of images from B, where the white arrow indicates the tracked structure. D. Average fluorescence intensity over time for endocytic structures with lifetimes within the following ranges: 10 to 20 s, 40 to 60 s, 60 to 80 s, and 80 to 100 s. Intensity shown for the liposome (Texas Red DHPE) and AP2 (JF646) channels. Cohorts were composed of 1647, 739, 563, and 368 events, for the 10-20s, 40-60s, 60-80s, and 80-100s cohorts, respectively. E-G. Distribution of clathrin-coated structure lifetimes for cells exposed to liposomes containing: 0 mol% (E), 10 mol% (F), and 20 mol% (G) biotinylated lipids. The insets of parts E-G compare median lifetimes of the clathrin structures that were associated with a liposome to those that did not. Error bars represent the standard error of the mean of N = 3 independent trials. Total number of clathrin-mediated endocytic events per graph was 8,804, 22,869, and 10,930, respectively. H. Cumulative probability of endocytic structure departure as a function of time for the data shown in G.

Figure 3.. Targeting does not significantly impact the overall probability that a liposome will be internalized by a clathrin-coated structure.

A. Bar graph of the number per area of liposomes that associated with a clathrin-coated structure for liposomes containing 0, 10, and 20 mol% of biotinylated lipids (t-test 0% to 10%; p = 0.070; n = 66, 84; t-test 0% to 20%; p = 0.523; n = 66, 68; t-test 10% to 20%; p = 0.256; n = 84, 68). B. Bar graph of the number per area of liposomes that were in internalized by a clathrin-coated structure for liposomes containing 0, 10, and 20 mol% of biotinylated lipids (t-test 0% to 10%; p = 0.082; n = 66, 84; t-test 0% to 20%; p = 0.048; n = 66, 68; t-test 10% to 20%; p = 0.722; n = 84, 68). C) Bar graph of the number per area of total liposomes beneath cells for liposomes containing 0, 10, and 20 mol% biotinylated lipids (t-test 0% to 10%; p = 0.015; n = 66, 84; t-test 0% to 20%; p = 0.417; n = 66, 68; t-test 10% to 20%; p = 0.124; n = 84, 68). For A-C, three independent trials were acquired for each condition with a minimum of 15 cells imaged per trial. Cells across trials were combined for a total of 66, 84, and 68 cellular crops exposed to liposomes containing 0, 10, and 20 mol% biotinylated lipids, respectively. Error bars represent the standard error of the mean. Significance between conditions was identified using a two-tailed student’s t-test with α = 0.05. D. Distribution of liposome diameters. The black curve is the overall size distribution of liposomes prior to their exposure to cells (47,502 liposomes). The red, gold, and blue curves are the size distributions for liposomes that penetrate beneath cells, for liposomes containing 20, 10, or 0 mol% biotinylated lipids. The red, gold, and blue distributions contain 56,130, 61,002, and 31,150 liposomes respectively.

Figure 4.. Targeting increases the probability of endocytic uptake for liposomes of intermediate diameter.

A-D. Distribution of liposome diameters for liposomes containing 0% or 20% biotinylated lipids. The top plot (black curve) is the overall distribution of liposome diameters (n = 47,502), repeated from Figure 3. The second plot is the distribution of diameters for liposomes that penetrated beneath cells (repeated from Figure 3). The third plot is the distribution of diameter for liposomes that penetrated beneath cells and associated with a clathrin-coated structure (12,282 (red) and 16,821 (blue) liposomes). The fourth plot is the distribution of diameters for liposomes that penetrated beneath cells, associated with a clathrin-coated structure, and became internalized (1,269 (red) and 1,577 (blue) liposomes). E. Bar graph representing the probability that a liposomes will associated with a clathrin-coated structure for the full distribution of liposome diameters (hashed bars) and the population of liposomes with diameters below 40 nm (solid bars), for liposomes containing 0 (blue), 10 (gold), or 20 (red) mol% biotinylated lipids (t-test 0% to 10%; p = 0.042; n = 66, 84; t-test 0% to 20%; p < 1x10⁻⁴; n = 66, 68; t-test 10% to 20%; p = 0.028). F. Bar graph representing the probability that a liposomes will be internalized by a clathrin-coated structure for the full distribution of liposome diameters (hashed bars) and the population of liposomes with diameters below 40 nm (solid bars), for liposomes containing 0 (blue), 10 (gold), or 20 (red) mol% biotinylated lipids (t-test 0% to 10%; p = 0.133; n = 66, 84; t-test 0% to 20%; p = 0.001; n = 66, 68; t-test 10% to 20%; p = 0.115). For E and F, N = 3 independent trials were run for each condition, with at least 15 cells imaged per trial. The total number of cellular crops from all trials in E-F was 66, 84, and 68 for liposomes containing 0, 10, or 20 mol% biotinylated lipids, respectively. Error bars represent the standard error of the mean for each population. Significance between conditions was identified using a two-tailed student’s t-test with α = 0.05. G. The ratio of the probability of internalization for liposomes containing 20 versus 0 mol % biotinylated lipids, plotted as a function of the liposome diameter cutoff. The average ratio for the entire population of liposome diameters is shown in orange. The significance of the differences between the data for each cutoff (black) and the population average (orange) were determined using a z-test (two-sample for means; 30 nm p = 0.748; 32.5 nm p = 0.055; 35 nm p < 1x10⁻⁵; 40 nm p < 1x10⁻⁵; 45 nm p < 1x10⁻⁵; 50 nm p < 1x10⁻⁵; 55 nm p = 0.000393; 60 nm p = 0.371; n = 82, n = 82). H. The ratio of the probability of liposome internalization for liposomes with diameters below a specific cutoff (horizontal axis) relative to the overall probability of internalization for the full population of liposomes (all diameters). The significance of differences between the data for liposomes containing 0 (blue) and 20 mol% (red) biotinylated lipids was determined using a z-test (two-sample for means; 30 nm p =0.0506; 35 nm p < 1x10⁻⁵; 40 nm p < 1x10⁻⁵; 45 nm p < 1x10⁻⁵; 50 nm p < 1x10⁻⁵; 55 nm 0.000868; 60 nm p = 0.422; n = 82, n = 82). The total number of cells in G and H were 82 and 82, respectively. Error bars represent the standard deviation.

Figure 5.

Schematic showing the ability of larger liposomes to penetrate beneath the basolateral cellular membrane due to targeting. In contrast, small liposomes have a high probability of penetration and internalization, regardless of targeting.