Boosting of the enhanced permeability and retention effect with nanocapsules improves the therapeutic effects of cetuximab

Abstract

Objective: The introduction of therapeutic antibodies (tAbs) into clinical practice has revolutionized tumor treatment strategies, but their tumor therapy efficiency is still far below expectations because of the rapid degradation and limited tumor accumulation of tAbs. Methods: We developed a nanocapsule-based delivery system to induce the self-augmentation of the enhanced permeability and retention (EPR) effect. This system constantly penetrated across the blood-tumor barrier into the tumor while avoiding the attack of tAbs by the immune system. The biodistribution and therapeutic effect were tested with single dose administration of nanocapsule-tAbs in vivo. Results: The accumulation of Nano(cetuximab) within subcutaneous PC9 tumors was gradually enhanced over 6 days after single dose administration, which was contrary to the biodistribution of native cetuximab. Nano(cetuximab) accumulated in tumor tissues via the EPR effect and released cetuximab. The released cetuximab acted on vascular endothelial cells to destroy the blood-tumor barrier and induce self-augmentation of the EPR effect, which in turn contributed to further tumor accumulation of long-circulating Nano(cetuximab). Compared with single dose administration of native cetuximab, Nano(cetuximab) showed an effective tumor suppressive effect for 3 weeks. Conclusions: The nanocapsule-based delivery system efficiently delivered tAbs to tumor tissues and released them to boost the EPR effect, which facilitated further tumor accumulation of the tAbs. This novel self-augmentation of the EPR effect facilitated by the biological characteristics of tAbs and nanotechnology contributed to the improvement of the therapeutic effect of tAbs, and stimulated new ideas for antibody-based tumor therapy.

Keywords: EPR effect; Endothelial cells; nanocapsule; single dose administration; therapeutic antibody.

Figure 1

(A) The preparation of therapeutic antibody tAb nanocapsules. (B) A representative transmission electron microscopy image of Nano(cetuximab). Scale bar: 100 μm. (C) The results of energy spectrum analysis of native cetuximab and Nano(cetuximab). (D) The size distributions of cetuximab and Nano(cetuximab). (E) Fluorescein isothiocyanate results of Nano(cetuximab). (F) Agarose gel electrophoresis results of Nano(cetuximab) after treatment with glutathione and (G) Western blot analysis of the activity of native cetuximab and Nano(cetuximab). (H) RTCA (Real Time Cellular Analysis) showing the cell proliferation profile after incubation with various groups.

Figure 2

(A) Cy5.5-labeled cetuximab incubated with macrophages for 0, 1, 2 and 4 h and imaged by confocal scanning microscopy; green represents actin, and red represents cetuximab, Scale bar: 40 μm. (B) Quantitation of the fluorescence intensity of the uptake of native cetuximab and Nano(cetuximab) by macrophages. (C) Blood clearance profiles of native cetuximab and Nano(cetuximab) in healthy female Kunming mice. (D) Representative fluorescence images of tumor-bearing mice at 1, 2, and 6 days after treatment with Cy5.5-labeled native cetuximab or Nano(cetuximab). (E) Radiant efficiencies of Cy5.5-labeled native cetuximab and Nano(cetuximab) in the subcutaneous tumor model. (F) Representative ex vivo fluorescence images of major organs. (G) Accumulation of Cy5.5-labeled native cetuximab and Nano(cetuximab) in tumor sections evaluated using a fluorescence microscope; green represents ERG, and red represents cetuximab. Scale bar: 100 μm. The significance level is shown as ^*P < 0.01.

Figure 3

(A) Electron microscopy analysis of the vasculature of tumors at 1 and 6 days after the administration of native cetuximab and Nano(cetuximab). Orange represents the vessel lumen, blue represents tumor cells, and purple represents apoptotic tumor cells. Scale bar: 4 μm. (B) A potential schematic illustration of the working mechanism of Nano(cetuximab).

Figure 4

(A) Confocal scanning microscopy images of frozen sections. Green represents EGFR, and red represents cetuximab. Scale bar: 100 μm. (B) Hematoxylin and eosin staining of the intestine. Scale bar: 400 μm.

Figure 5

(A) Photographs of tumors after treatment on day 21. (B) The tumor volume was measured every 2 days. The significance levels are shown as ^*P < 0.01. (C) Representative photomicrographs showing hematoxylin and eosin staining. Scale bar: 200 μm. (D) Immunohistochemical staining for p-EGFR, p-AKT, Ki67, and CD31 in tissue samples. Scale bar: 50 μm.