Cytoplasmic delivery of liposomes into MCF-7 breast cancer cells mediated by cell-specific phage fusion coat protein

Abstract

Earlier, we have shown that doxorubicin-loaded liposomes (Doxil) modified with a chimeric phage fusion coat protein specific toward MCF-7 breast cancer cells identified from a phage landscape library demonstrated a significantly enhanced association with target cells and an increased cytotoxicity. Based on some structural similarities between the N-terminus of the phage potein and known fusogenic peptides, we hypothesized that, in addition to the specific targeting, the phage protein may possess endosome-escaping potential and an increased cytotoxicity of drug-loaded phage protein-targeted liposomes may be explained by an advantageous combination of both, cell targeting and endosomal escape of drug-loaded nanocarrier. The use of the fluorescence resonance energy transfer (FRET) technique allowed us to clearly demonstrate the pH-dependent membrane fusion activity of the phage protein. Endosomal escape and cytosolic delivery of phage-liposomes was visualized with fluorescence microscopy. Endosome acidification inhibition by bafilomycin A 1 resulted in decreased cytotoxicity of the phage-Doxil, while the endosome disruption by chloroquine had a negligible effect on efficacy of phage-Doxil, confirming its endosomal escape. Our results demonstrated an endosome-escaping property of the phage protein and provided an insight on mechanism of the enhanced cytotoxicity of phage-Doxil.

Figure 1. Endocytic uptake of phage-liposomes

A) shows the effect of endosome acidification on phage-Doxil-mediated cytotoxicity as revealed by Bafilomycin A1 inhibition.(*p<0.05; n=5, mean ± SEM). B) Fluorescence microscopy shows that rhodamine-labeled phage liposomes (red) co-localized with transferrin-labeled early endosomes (green).

Figure 2. Membrane fusion activity of phage protein detected by FRET

(A, B) pH-dependent membrane fusion by phage protein.( – double-labeled liposomes only; – double-labeled liposomes + plain liposomes; – double-labeled liposomes + 1% phage-liposomes); (C, D) Effect of dose of phage protein on membrane fusion at acidic pH.(– double-labeled liposomes only; – double labeled liposomes + 1% phage-liposomes; – double-labeled liposomes + 0.5% phage-liposomes; – double-labeled liposomes + 0.05% phage-liposomes). (E) Time course of the pH- and dose-dependent membrane fusion mediated by the phage protein. (n=3; mean ± SD).

Figure 3. Intracellular membrane fusion and endosomal escape potential of phage protein

The inhibition of the endosomal acidification by NH₄Cl blocks phage protein–induced intracellular membrane fusion detected by FRET. Black bar: 1 h binding at 4 °C followed by 1 h internalization at 37 °C; Grey bar: 1 h binding at 4 °C followed by 1 h internalization at 37 °C with NH₄Cl ; White bar: 1 h binding at 4 °C . (*p<0.05; n=3, mean ± SD).

Figure 4. Endosome release of rhodamine-labeled phage-liposomes

A) Perinuclear punctuate pattern of plain liposomes in the absence of the endosome acidification inhibitor - NH₄Cl, suggesting their entrapment into endosomes or lysosomes. B) Punctuate pattern of plain liposomes in the presence of the endosome acidification inhibitor - NH₄Cl, C) Diffuse subcellular pattern of phage-liposomes, indicating their subcellular locations in cytosol; D) Perinuclear punctuate pattern, indicating NH₄Cl inhibition on endosome release of phage-liposomes.

Figure 5. Cytosolic delivery of HPTS encapsulated by phage-liposomes

While much less green fluorescence is observed in the case of plain liposomes, phage-liposome treatment shows strong fluorescence emission at the 494nm excitation, indicating that HPTS is released into the neutral cytosol.

Figure 6. Endosomal escape of phage-Doxil

Endosome disruption by chloroquine showing enhanced cytotoxicity of Doxil but a negligible effect on phage-Doxil. (*p<0.05; n=6, Mean±SEM).