Paclitaxel-loaded PEG-PE-based micellar nanopreparations targeted with tumor-specific landscape phage fusion protein enhance apoptosis and efficiently reduce tumors

Abstract

In an effort to improve the therapeutic index of cancer chemotherapy, we developed an advanced nanopreparation based on the combination of landscape phage display to obtain new targeting ligands with micellar nanoparticles for tumor targeting of water-insoluble neoplastic agents. With paclitaxel as a drug, this self-assembled nanopreparation composed of MCF-7-specific phage protein and polyethylene glycol-phosphatidylethanolamine (PEG-PE) micelles showed selective toxicity to target cancer cells rather than nontarget, non cancer cells in vitro. In vivo, the targeted phage micelles triggered a dramatic tumor reduction and extensive necrosis as a result of improved tumor delivery of paclitaxel. The enhanced anticancer effect was also verified by an enhanced apoptosis and reduced tumor cell proliferation following the treatment with the targeted micellar paclitaxel both in vitro and in vivo. The absence of hepatotoxicity and pathologic changes in tissue sections of vital organs, together with maintenance of overall health of mice following the treatment, further support its translational potential as an effective and safe chemotherapy for improved breast cancer treatment.

Figure 1. Characterization of micellar paclitaxel (PCT)

Dynamic light scattering analysis of size distribution of MCF-7-targeted PCT phage micelles (A) and non-targeted PCT micelles (B); (C) TEM image of the targeted phage micelles; (D) Zeta-potential; (E) Storage stability at 4°C.

Figure 2. In vitro antitumor activity of MCF-7 targeted PCT phage micelles

Following the treatment with targeted and non-targeted micellar PCT, (A-B) cytotoxicity of MCF-7 cells and of C166 cells; (C) FACS analysis of Annexin V conjugates and 7-ADD co-staining MCF-7 cells; (D) FACS analysis of immunofluorescent staining of MCF-7 cells with BrdU incorporation coupled with 7-ADD staining for the determination of cells with active DNA synthesis and cell cycle position. Mean ± SEM, n= 3, p<0.05.

Figure 3. In vivo antitumor activity following the treatment with micellar PCT in MCF-7 tumor-bearing nude mice

(A) Estimation of tumor volume using a caliper. Tumor volume from day 0 of treatment (%) = [(Tumor volume at days after treatment) / (Tumor volume at treatment day 0)] ×100; (B) Final tumor weight; (C) A representative set of regular MR images of tumors untreated and treated with non-targeted PCT micelles and MCF-7 targeted PCT phage-micelles at the endpoint of treatment; (D) Estimation of tumor volume using MR images; (E) Tumor growth inhibition (%) estimated by MRI, 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. Mean ± SEM, n= 5 * p<0.05 or p<0.005.

Figure 4. Ex vivo antitumor activity following the treatment with micellar PCT in MCF-7 tumor-bearing nude mice

(A) Representative images of H & E staining of tumor sections. Necrotic cells showing eosinophilic cytosol (pink) accompanied by the absence of hemotoxylin-stained nuclei (blue); viable cells showing eosinophilic cytosol (pink) accompanied by hemotoxylin-stained nuclei (blue); (B) Representative images of tumor sections with TUNEL staining, showing enhanced in vivo apoptosis by the treatment with MCF-7-targeted PCT phage-micelles; (C) Apoptosis index (%), defined as the percentage of TUNEL- positive nuclei of a total of 2000 ± 100 tumor cells by manual counting; (D) Representative images of tumor sections with immunohistochemical staining for the Ki-67 proliferation marker antigens, showing enhanced anti-proliferation by the treatment with MCF-7-targeted PCT- phage-micelles; (E) Proliferation Index (%) defined as the percentage of Ki67-positive nuclei of a total of 2000 ±100 tumor cells by manual counting. Mean ± SEM, n= 5, p<0.05 or p<0.001.

Figure 5. Enhanced tumor delivery of PCT by MCF-7-targeted PCT phage-micelles

(A) Quantification of tumor-associated PCT expressed as ng PCT per g of tumor tissue; (B) PCT Tumor-to-RES ratio. A one-way ANOVA was followed by LSD post hoc tests to analyze the statistic. Mean ± SEM, n= 3, p<0.05.

Figure 6. Evaluation of potential side-effects of micellar PCT

(A) Mouse body weight following treatment; (B) The effect of treatment on liver enzyme activity of mice; (C) Histological examination of tissue sections of vital organs following the treatment. Mean ± SEM, n=3.