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Comparative Study
. 2012:7:4223-37.
doi: 10.2147/IJN.S34105. Epub 2012 Aug 1.

Biodistribution and endocytosis of ICAM-1-targeting antibodies versus nanocarriers in the gastrointestinal tract in mice

Viraj Mane  1 , Silvia Muro
Affiliations
Comparative Study

Biodistribution and endocytosis of ICAM-1-targeting antibodies versus nanocarriers in the gastrointestinal tract in mice

Viraj Mane et al. Int J Nanomedicine. 2012.

Abstract

Drug delivery to the gastrointestinal (GI) tract is key for improving treatment of GI maladies, developing oral vaccines, and facilitating drug transport into circulation. However, delivery of formulations to the GI tract is hindered by pH changes, degradative enzymes, mucus, and peristalsis, leading to poor GI retention. Targeting may prolong residence of therapeutics in the GI tract and enhance their interaction with this tissue, improving such aspects. We evaluated nanocarrier (NC) and ligand-mediated targeting in the GI tract following gastric gavage in mice. We compared GI biodistribution, degradation, and endocytosis between control antibodies and antibodies targeting the cell surface determinant intercellular adhesion molecule 1 (ICAM-1), expressed on GI epithelium and other cell types. These antibodies were administered either as free entities or coated onto polymer NCs. Fluorescence and radioisotope tracing showed proximal accumulation, with preferential retention in the stomach, jejunum, and ileum; and minimal presence in the duodenum, cecum, and colon by 1 hour after administration. Upstream (gastric) retention was enhanced in NC formulations, with decreased downstream (jejunal) accumulation. Of the total dose delivered to the GI tract, ∼60% was susceptible to enzymatic (but not pH-mediated) degradation, verified both in vitro and in vivo. Attenuation of peristalsis by sedation increased upstream retention (stomach, duodenum, and jejunum). Conversely, alkaline NaHCO(3), which enhances GI transit by decreasing mucosal viscosity, favored downstream (ileal) passage. This suggests passive transit through the GI tract, governed by mucoadhesion and peristalsis. In contrast, both free anti-ICAM and anti-ICAM NCs demonstrated significantly enhanced upstream (stomach and duodenum) retention when compared to control IgG counterparts, suggesting GI targeting. This was validated by transmission electron microscopy and energy dispersive X-ray spectroscopy, which revealed anti-ICAM NCs in vesicular compartments within duodenal epithelial cells. These results will guide future work aimed at improving intraoral delivery of targeted therapeutics for the treatment of GI pathologies.

Keywords: ICAM-1 targeting; antibody; endocytosis; gastrointestinal tract; polymer nanocarriers.

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Figures

Figure 1
Figure 1
Biodistribution and degradation of IgG in the GI tract. Mice were orally gavaged with 125I-IgG in either PBS or NaHCO3. One hour later, the indicated sections of the GI were harvested and measured for their 125I-content, expressed as % ID (A). Alternatively, samples were subjected to TCA precipitation to determine the percentage of free 125Iodine, reflective of antibody degradation (B). Notes: Data are mean ± SEM (n ≥ 3). *P < 0.05 between saline and NaHCO3 groups. Abbreviations: PBS, phosphate-buffered saline; GI, gastrointestinal; TCA, trichloroacetic acid; % ID, percentage of the total injected dose; SEM, standard error of the mean.
Figure 2
Figure 2
In vitro degradation of IgG under GI-mimicking conditions. 125I-IgG was incubated for the indicated time periods in SGF with or without pepsin, or SIF with or without pancreatin, and the percentage of 125I-IgG degradation was calculated as described in Figure 1. Curves were fitted by software regression analysis. Note: Data are mean ± SEM (n ≥ 3 per experiment and at least two independent experiments). Abbreviations: SGF, simulated gastric fluid; SIF, simulated intestinal fluid; SEM, standard error of the mean.
Figure 3
Figure 3
Biodistribution and degradation of IgG nanocarriers in the GI tract. Mice were gavaged with 125I-IgG-coated model polymer NCs (IgG NCs) in PBS, final diameter 269.8 ± 6.3 nm, and compared to their 125I-IgG counterparts. One hour later, the indicated sections of the GI tract were harvested and measured for their 125I-content, expressed as % ID (A). Samples were also subjected to TCA precipitation to determine the percentage of free 125Iodine, reflective of degradation (B). Notes: Data are mean ± SEM (n ≥ 3). *P < 0.05 between IgG and IgG NC groups. Abbreviations: GI, gastrointestinal; NC, nanocarrier; PBS, phosphate-buffered saline; % ID, percentage of the total injected dose; TCA, trichloroacetic acid; SEM, standard error of the mean.
Figure 4
Figure 4
In vitro degradation of IgG nanocarriers under GI-mimicking conditions. 125I-IgG NCs were incubated for the indicated time periods in SGF ± pepsin or SIF ± pancreatin, followed by TCA precipitation to determine the percentage of free 125Iodine, reflective of degradation, as described in Figure 1. Curves were fitted by software regression analysis. Note: Data are mean ± SEM (n ≥ 3 per experiment and at least two independent experiments). Abbreviations: GI, gastrointestinal; NC, nanocarrier; SGF, simulated gastric fluid; SIF, simulated intestinal fluid; TCA, trichloroacetic acid; SEM, standard error of the mean.
Figure 5
Figure 5
Biodistribution of anti-ICAM and anti-ICAM nanocarriers in the GI tract. Mice were gavaged with PBS containing 125I-anti-ICAM vs 125I-IgG (A) or 125I-anti-ICAM NCs vs 125I-IgG NCs (B), and 125Iodine biodistribution in the stomach, duodenum, and distal GI regions (encompassing jejunum, ileum, cecum, and colon) was assessed one hour later as described in Figure 1. A comparison of the biodistribution of 125I-anti-ICAM vs 125I-anti-ICAM NCs is shown in (C). Notes: Results are expressed as % ID. Data are mean ± SEM, (n ≥ 3). (A) and (B) *P < 0.05; **P < 0.005 between nontargeting IgG and ICAM-targeting groups. (C) *P < 0.05 between anti-ICAM free antibody and anti-ICAM NCs. Abbreviations: ICAM, intercellular adhesion molecule; GI, gastrointestinal; PBS, phosphate-buffered saline; NC, nanocarrier; % ID, percentage of the total injected dose; SEM, standard error of the mean.
Figure 6
Figure 6
Visualization of anti-ICAM nanocarriers bound to GI tissue. Mice were gavaged with FITC-labeled anti-ICAM NCs in PBS, then euthanized after 15 minutes. Cross-sectional dissections were made in the stomach, duodenum, jejunum, and ileum. Notes: Dissected tissue was thoroughly rinsed to remove unbound NCs and imaged by microscopy to detect tissue-associated fluorescent NCs. Scale bar = 500 μm. Abbreviations: ICAM, intercellular adhesion molecule; GI, gastrointestinal; NC, nanocarrier; PBS, phosphate-buffered saline.
Figure 7
Figure 7
Visualization of epithelial endocytosis of anti-ICAM nanocarriers into GI tissue. Mice were gavaged with saline (A–C) or anti-ICAM-coated iron oxide nanoparticles suspended in NaHCO3 (D–I). Notes: GI tissue was isolated after 10 minutes, processed, and imaged by TEM and iron EDS. In each row, a lower-magnification TEM image of the duodenum (left) is followed by a higher-magnification TEM image (middle) and its corresponding EDS analysis (right). White boxes indicate regions of EDS analysis. Arrows indicate electron-dense vesicular structures while arrowheads indicate non-vesicular structures. Scale bar = 500 nm. Abbreviations: mv, microvilli; ec, enterocyte; ICAM, intercellular adhesion molecule; GI, gastrointestinal; TEM, transmission electron microscope; EDS, energy dispersive X-ray spectroscopy.
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