Lysyl oxidase engineered lipid nanovesicles for the treatment of triple negative breast cancer

Abstract

In the field of oncology research, a deeper understanding of tumor biology has shed light on the role of environmental conditions surrounding cancer cells. In this regard, targeting the tumor microenvironment has recently emerged as a new way to access this disease. In this work, a novel extracellular matrix (ECM)-targeting nanotherapeutic was engineered using a lipid-based nanoparticle chemically linked to an inhibitor of the ECM-related enzyme, lysyl oxidase 1 (LOX), that inhibits the crosslinking of elastin and collagen fibers. We demonstrated that, when the conjugated vesicles were loaded with the chemotherapeutic epirubicin, superior inhibition of triple negative breast cancer (TNBC) cell growth was observed both in vitro and in vivo. Moreover, in vivo results displayed prolonged survival, minimal cytotoxicity, and enhanced biocompatibility compared to free epirubicin and epirubicin-loaded nanoparticles. This all-in-one nano-based ECM-targeting chemotherapeutic may provide a key-enabling technology for the treatment of TNBC.

Figure 1

(A) Physical characterization of lipid-based nanocarriers. (B) Cytometry analysis of Lipo and Lipo-LOX formulations. (C) FTIR analysis of Lipo and Lipo-LOX formulations.

Figure 2

(A) Protein analysis of secreted LOX in monolayer cultures not treated and treated with all formulations, full-length blots/gels are presented in Supplementary Fig. 2A. (B) Protein analysis of secreted LOX in 3D cultures not treated and treated with all formulations, full-length blots/gels are presented in Supplementary Fig. 2B, C. (C) Confocal analysis of 3D cultures exposed to formulations encapsulated with anthracycline after 6 h from the exposure. Nuclei were stained with dapi (blue), actin filaments were stained with phalloidin (green), epirubicin (red) and merge. (D) Confocal analysis of 3D cultures exposed to formulations encapsulated with anthracycline after 48 h from the exposure. Scale bar 50 µm (E) Mean fluorescence intensity of anthracycline detected after 6 and 48 h. (F) Cell viability of tumor cells after treatment with all studied formulations in standard monolayer cultures, negative control is CTR and positive control is LIPO-EPI-LOX. (G) Cell viability of tumor cells after treatment with all studied formulations in 3D cultures, negative control is CTR and positive control is LIPO-EPI-LOX. *p < 0.05, **p < 0.01.

Figure 3

(A) In vivo biodistribution analysis of Lipo-EPI and Lipo-EPI-LOX in orthotropic xenograft mouse model of human TNBC at different time points (0, 1, 2 and 24 h post injections). (B) bioluminescence and fluorescence analysis of explanted organs (liver, spleen, lungs, heart, kidneys, tumors) to detect EPI localization. (C) Lipo-EPI and Lipo-EPI-LOX fluorescent signal quantification in tumor. (D) Lipo-EPI and Lipo-EPI-LOX fluorescent signal quantification in explanted organs.

Figure 4

(A) Tumor growth curves of orthotopic xenograft mouse model of human TNBC. Five group mice (n = 10 each group) treated with empty liposome (CTR), Lipo-LOX, LIPO-EPI, free EPI and Lipo-EPI-LOX. (B) Delta quantification of TNBC bioluminescent signal after 4 weeks of treatment. (C) Representative example of H&E stained sections of explanted tumors 55 days post treatment. Control: markedly atypical and pleomorphic cells (5% necrotic tumor cells). Scale bar is 400 µm. Lipo-LOX: fibrotic tissue with atypical cells and neoplastic crown (25% necrotic tumor cells). Lipo-EPI: Neoplastic cells and necrotic bands (50% necrotic tumor cells). Free EPI: tumor cells infiltrating adipose tissue with minimal necrotic features e limited inflammation (20% necrotic tumor cells). Lipo-EPI-LOX : tumor cells infiltrating adipose tissue with artifacts (60% necrotic tumor cells). Representative example of collagen stained sections of explanted tumors 55 days post treatment. Control: collagen fibers linearization ˃ 200 µm, Lipo-LOX collagen fibers linearization ˂ 200 µm, Lipo-EPI collagen fibers linearization ˃ 200 µm, free EPI collagen fibers linearization ˃ 200 µm, Lipo-EPI-LOX collagen fibers linearization ˂ 200 µm. Scale bar is 400 µm. (D) Kaplan–Meier curve indicating survival in tumor-bearing mice after tumor induction in the listed treatment groups (Control, Lipo-LOX, LIPO-EPI, free EPI and Lipo-EPI-LOX).

Figure 5

(A) body weight change percentage of mice at the end of the study. (B) Representative example of H&E stained sections of explanted hearts 55 days post treatment. Control: normal cardiac tissue. Lipo-LOX: no increase of inflammatory infiltrate. Lipo-EPI: minimal focal increase of inflammatory infiltrate (granulocytes and lymphocytes), artifacts. Free EPI: increased cellularity due to possible inflammatory infiltrate. Lipo-EPI-LOX : no significant increment of lymphoid infiltrate, artifacts. Scale bar is 400 µm. Representative example of H&E stained sections of explanted lungs 55 days post treatment. Control: phlogosis 70%. Lipo-LOX: phlogosis 30%. Lipo-EPI: phlogosis 15%. Free EPI: phlogosis 15%. Lipo-EPI-LOX : phlogosis 5%. Scale bar is 400 µm.