Enhanced tumor delivery and antitumor activity in vivo of liposomal doxorubicin modified with MCF-7-specific phage fusion protein

Abstract

A novel strategy to improve the therapeutic index of chemotherapy has been developed by the integration of nanotechnology with phage technique. The objective of this study was to combine phage display, identifying tumor-targeting ligands, with a liposomal nanocarrier for targeted delivery of doxorubicin. Following the proof of concept in cell-based experiments, this study focused on in vivo assessment of antitumor activity and potential side-effects of phage fusion protein-modified liposomal doxorubicin. MCF-7-targeted phage-Doxil treatments led to greater tumor remission and faster onset of antitumor activity than the treatments with non-targeted formulations. The enhanced anticancer effect induced by the targeted phage-Doxil correlated with an improved tumor accumulation of doxorubicin. Tumor sections consistently revealed enhanced apoptosis, reduced proliferation activity and extensive necrosis. Phage-Doxil-treated mice did not show any sign of hepatotoxicity and maintained overall health. Therefore, MCF-7-targeted phage-Doxil seems to be an active and tolerable chemotherapy for breast cancer treatment.

From the clinical editor: The authors of this study successfully combined phage display with a liposomal nanocarrier for targeted delivery of doxorubicin using MCF-7-targeted phage-Doxil nanocarriers in a rodent model. The method demonstrated improved efficiency and reduced hepatotoxicity, paving the way to future clinical trials addressing breast cancer.

Keywords: Breast cancer targeting; Cancer nanomedicines; Drug delivery; Liposomes; Phage display.

Figure 1. Characterization of LipoDox and phage-Doxil

(A) Size and size distribution of LipoDox and Phage-Doxil measured by dynamic light scattering. (B) LipoDox and phage-Doxil elution profiles during SEC-HPLC. (C) Morphology of targeted phage-Doxil observed by transmission electron microscopy. (D) Zeta-potential of LipoDox and phage-Doxil. (E) Storage stability of LipoDox and phage-Doxil at 4°C. (F) Serum stability of LipoDox and phage-Doxil incubated in 50% FBS at 37°C.

Figure 2. Antitumor activity of Doxil and phage-Doxil

(A) Tumor volume was estimated using a caliper. Tumor volume changes following the treatment in (A1) subcutaneous and (A2)orthotopic MCF-7 tumor-bearing nude mice. % Tumor volume from day 0 = (Tumor volume_{after treatment}) / (Tumor volume _{at day 0}) ×100; (B) Determination of tumor volume at the endpoint by MRI imaging with subcutaneous xenografts. (B1) The representative set of regular MRI images of tumors untreated and treated with Doxil, non-targeted phage-Doxil and MCF-7 targeted phage-Doxil. (B2) Tumor volume. (B3) % Tumor growth inhibition, defined as the difference between the tumor volume of the untreated group and the tumor volume of the treated group divided by the tumor volume in untreated group ×100. A one-way ANOVA was followed by LSD post hoc tests. Mean ± SEM, n=3-5. * p<0.05; ** p<0.005.

Figure 3. The confirmation of antitumor activity by H & E staining

Representative images of tumor sections. Necrotic cells (N) showing eosinophillic cytosol (pink) accompanied by the absence of hemotoxylin-stained nuclei (blue); viable cells (V) showing eosinophillic cytosol (pink) accompanied by hemotoxylin-stained nuclei (blue).

Figure 4. Enhancement in tumor delivery of the drug, apoptosis, and anti-proliferative activity by MCF-7 targeted phage-Doxil

(A) Tumor accumulation of doxorubicin observed by fluorescence microscopy, Red fluorescence: Doxorubicin; Blue fluorescence: DAPI-staining nuclei. (B) Quantification of doxorubicin tumor deposition expressed by ng doxorubicin per g of tumor tissue. (C) Tumor-to-muscle ratio of doxorubicin. (D) TUNEL staining of tumor sections. (E) Immunohistochemical staining of the Ki-67 proliferation marker on the tumor sections.

Figure 5. Evaluation of potential side-effects of Doxil (or LipoDox) and Phage-Doxil

(A) Body weight of mice following treatment in subcutaneous (A1), and orthotopic MCF-7 xenografts (A2). (B) Histology examination of tissue sections of vital organs following treatment. (C) The effect of treatment on liver enzyme activity of mice. AST: Aspartate Aminotransaminase Activity; ALT: Alanine Transaminase Activity. A one-way ANOVA was followed by LSD post hoc test to analyze the statistic. Mean ± SEM, n=3-5. * p >0.05.