Nanoparticle uptake by circulating leukocytes: A major barrier to tumor delivery

Abstract

Decades of research into improving drug delivery to tumors has documented uptake of particulate delivery systems by resident macrophages in the lung, liver, and spleen, and correlated short circulation times with reduced tumor accumulation. An implicit assumption in these studies is that nanoparticles present in the blood are available for distribution to the tumor. This study documents significant levels of lipoplex uptake by circulating leukocytes, and its effect on distribution to the tumor and other organs. In agreement with previous studies, PEGylation dramatically extends circulation times and enhances tumor delivery. However, our studies suggest that this relationship is not straightforward, and that particle sequestration by leukocytes can significantly alter biodistribution, especially with non-PEGylated nanoparticle formulations. We conclude that leukocyte uptake should be considered in biodistribution studies, and that delivery to these circulating cells may present opportunities for treating viral infections and leukemia.

Keywords: Gene delivery; Leukocyte uptake; Lipoplexes; PEGylation.

Figure 1.

Analysis of lipoplex uptake by blood cells via flow cytometry. Balb/c mice (n = 3) bearing CT26 tumors were intravenously injected with PicoGreen®-labelled lipoplexes, and blood was harvested after 1 h. Blood cells were stained with an antibody against CD45 which binds to leukocytes. The percentage of CD45− and CD45+ cells that are positive for PicoGreen® staining (A), and the staining intensity of CD45+ (B) and CD45− (C) are depicted. Note that PicoGreen®staining is largely confined to leukocytes (CD45+), with minimal staining of erythrocytes (CD45−). In addition, the intensity of PicoGreen® staining is much higher in leukocytes.

Figure 2.

Analysis of lipoplex uptake by leukocytes via flow cytometry. Balb/c mice bearing CT26 tumors were intravenously injected with carboxyfluorescein-labelled lipoplexes, and blood was harvested after 1 h. CD45+ leukocytes were stained with antibodies to quantify uptake by myeloid cells (CD45+, CD11b+, CD335−), NK cells (CD45+, CD11b+, CD335+), CD8+ T cells (CD45+,CD8+,CD4−, CD11b−), and CD4+ T cells (CD45+,CD4+,CD8−,CD11b−). The percent of each cell population that was particle positive is depicted. Bars represent the mean and standard error of blood isolated from 3 mice.

Figure 3.

Time dependence of lipoplex distribution. Plasmid levels in both the plasma and the cell fraction of the blood (A) and corresponding levels in the tumor (B) are shown at 1 and 24 h. Consistent with the low levels of plasmid, expression (24 h) in the tumor was minimal (C). Bars represent the mean and standard error of samples harvested from 3 mice at each timepoint.

Figure 4.

Biodistribution and expression in organs. Plasmid levels decreased in all organs between 1 and 24 h (A). Expression at 24 h (B) in organs does not correlate with plasmid levels. Bars represent the mean and standard error of organs harvested from 3 mice at each timepoint.

Figure 5.

Leukocyte uptake of PEGylated lipoplexes. Uptake of PEGylated lipoplexes by CD45+ cells (A) decreases between 1 and 24 h. Uptake of PEGylated lipoplexes by specific cell types at 1 and 24 h (B): myeloid cells (CD45+, CD11b+, CD335−), NK cells (CD45+, CD11b+, CD335+), CD8+ T cells (CD45+,CD8+,CD4−,CD11b−), and CD4+ T cells (CD45+,CD4+,CD8−,CD11b−). The percent of each cell population that was particle positive is depicted. Bars represent the mean and standard error of blood isolated from 3 mice.

Figure 6.

Time dependence of PEGylated lipoplex distribution. Plasmid levels in the plasma were much higher than the cell fraction (A), and corresponded with higher tumor levels (B) as compared to non-PEGylated lipoplexes. Note that plasmid levels in the tumor increase from 1 h to 24 h, consistent with the longer half-life (≈ 3 h) of the PEGylated formulation. Bars represent the mean and standard error of tissues harvested from 3 mice at each timepoint.

Figure 7.

Biodistribution of PEGylated lipoplexes. Plasmid levels increased at 24 h in both liver and spleen (A). Expression (24 h) was highest in liver and spleen, consistent with the greater plasmid accumulation (B). Bars represent the mean and standard error of organs harvested from 3 mice at each timepoint.

Figure 8.

Biodistribution of lipoplex formulations in SCID mice. Plasmid accumulation was greatest in the liver, and accumulation in tumor, lung, and spleen were comparable (A). Expression (24 h) was highest in liver, consistent with the greater plasmid accumulation (B). Bars represent the mean and standard error of organs harvested from 3 mice at each timepoint.

Figure 9.

Blood circulation. Plasmid levels in the plasma and cell fraction were quantified separately at different time points for non-PEGylated (A) and PEGylated (B) lipoplexes in Balb/c mice, and non-PEGylated lipoplexes in SCID mice (C). Note the much greater levels of PEGylated lipoplexes in the plasma; calculated half-lives are presented in Table 2. Each symbol represents the mean and standard error of blood samples from 3 mice.