Synergistic antitumor activity from two-stage delivery of targeted toxins and endosome-disrupting nanoparticles

Abstract

Plant-derived Type I toxins are candidate anticancer therapeutics requiring cytosolic delivery into tumor cells. We tested a concept for two-stage delivery, whereby tumor cells precoated with an antibody-targeted gelonin toxin were killed by exposure to endosome-disrupting polymer nanoparticles. Co-internalization of particles and tumor cell-bound gelonin led to cytosolic delivery and >50-fold enhancement of toxin efficacy. This approach allows the extreme potency of gelonin to be focused on tumors with significantly reduced potential for off-target toxicity.

Figure 1

Schematic of temporally staggered, staged delivery of a tumor-targeted toxin and a cytosolic delivery chaperone.

Figure 2

Structure and composition of PEGylated lipid-coated PBAE particles.

Figure 3

pH-responsive lipid-enveloped PBAE particles disrupt endosomes and deliver coendocytosed calcein into the cytosol and nucleus of tumor cells. B16F10 cells were incubated for 1 h at 37 °C with calcein alone or calcein and rhodamine-labeled lipid-coated PBAE particles, washed to remove unbound particles, then imaged live by confocal microscopy. Representative confocal images of B16F10 melanoma cells incubated with calcein (green) alone (A) or coincubated with calcein and lipid-coated PBAE particles (red, B = brightfield-calcein-particle fluorescence overlay, C = brightfield-particle fluorescence overlay). Scale bars 20 μm.

Figure 4

Effect of molecular weight and charge of polar cargo macromolecules on cytosolic delivery through coendocytosis with lipid-coated PBAE particles. (A) B16F10 cells were incubated with anionic or neutral fluorescent dextran conjugates (red, 150 μg/mL) of various molecular weights, with or without lipid-coated PBAE nanoparticles (unlabeled, 75 μg/mL) for 1 h at 37 °C. Cells were washed and imaged live via confocal microscopy. Scale bars 20 μm. (B) Plot of mean fluorescence intensity detected in cells computed from replicate fields of view for each dextran relative to cotreatment with particles (***, p < 0.0001). (C) Binding of various dextrans to nanoparticles coincubated in DMEM containing 10% serum for 18 h at 37 °C at concentrations identical to the conditions of A and B.

Figure 5

Cytosolic delivery of soluble phalloidin by lipid-coated PBAE particles. Confocal images of B16F10 cells incubated with (A) 10 μM or (D) 25 μM phalloidin alone or coincubated with phalloidin and 75 μg/mL rhodamine-labeled lipid-coated PBAE particles (B–C,E). (A,B,D,E = brightfield-phalloidin fluorescence overlays; C = magnified, phalloidin-particle fluorescence image of boxed cell in B; red, nanoparticles; green, phalloidin-alexa 488 conjugate). (F) Cytotoxicity of B16F10 cells treated with various concentrations of phalloidin alone or combined with 50 or 75 μg/mL particles for 24 h. (***, p < 0.0001).

Figure 6

Killing of tumor cells by particle-chaperoned immunotoxins. (A–D) Tumor cell lines A431 (EGFR-expressing, A,B) or B16F10 (EGFR-negative, C,D) were incubated with E4rGel immunotoxin and/or lipid-coated PBAE particles at the indicated concentrations for 24 h and then washed into fresh medium. Viability was measured at 72 h via the WST-1 metabolic assay (A,C) and used to compute the combination index where a value >1 indicates synergistic tumor cell killing in the combination treatment (particles + E4rGel) compared to immunotoxin alone (B,D). (E–F) The normalized metabolic rate (E) and combination index (F) for MC38 tumor cells or MC38 cells expressing CEA following incubation with 1 nM of the CEA-targeted C7rGel immunotoxin and lipid-coated PBAE particles at the indicated concentrations. (A: ***, p < 0.0001; B,D: ***, p < 0.0001, **, p < 0.01 relative to combination index = 1).