Targeted Nutlin-3A Loaded Nanoparticles Inhibiting P53–MDM2 Interaction: Novel Strategy for Breast Cancer Therapy

Abstract

Aim: The objective of the present study is to prepare and characterize nutlin-3a loaded polymeric poly(lactide-co-glycolide) nanoparticles (NPs) surface functionalized with transferrin ligand, to deliver the encapsulated drug in a targeted manner to its site of action and to evaluate the efficacy of the nanoformulation in terms of its cellular uptake, cell cytotoxicity, cell cycle arrest, apoptosis and activation of p53 pathway at molecular level in MCF-7 breast cancer cell line. Method: Nutlin-3a loaded poly(lactide-co-glycolide) NPs were prepared following the single oil-in-water emulsion method. Physicochemical characterization of the formulation included size and surface charge measurement, transmission electron microscopy characterization, study of surface morphology using scanning electron microscopy, Fourier-transform infrared spectral analysis and in vitro release kinetics studies. Furthermore, targeting ability of the conjugated system was assessed by cellular uptake and cell cytotoxicity studies in an in vitro cell model. Molecular basis of nutlin-3a-mediated p53 activation pathway was investigated by western blot analysis. Inhibition of cell cycle progression and apoptosis was evaluated by flow cytometry. Results: Physiochemical characterization of the formulations revealed that nutlin-3a was efficiently encapsulated in the nanoparticulate system, reaching an encapsulation efficiency of approximately 80% with size of approximately 220 nm and negative zeta potential of approximately -10.4 mV. Higher cellular uptake efficiency of the conjugated system proved the effectiveness of targeted therapy. IC₅₀ values, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay, showed superior antiproliferative activity of transferrin-conjugated NPs over unconjugated NPs and native nutlin-3a, owing to enhanced cellular uptake by cancer cells. At the molecular level the conjugated system showed enhanced activation of p53 pathway in comparison to native drug as evident from western blot analysis. Augmented cell cycle arrest and apoptosis was exhibited by the conjugated system. Thus, our results suggest that transferrin-conjugated nutlin-3a loaded NPs could be a potential drug carrier system for targeted delivery of potent anticancer drug nutlin-3a for breast cancer therapy.

Keywords::

• cancer therapy
• MDM2 inhibition
• nanoparticle
• nutlin-3a
• surface functionalization
• targeted therapy

Financial & competing interests disclosure

This work was supported by the Department of Science and Technology, Government of India (grant no. INT/AUS/P-8/06). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Acknowledgements

The authors would like to thank Dr S Devdas, Scientist, Institute of Life Sciences, Bhubaneswar, for his help in flow cytometry experiments.

Additional information

Funding

This work was supported by the Department of Science and Technology, Government of India (grant no. INT/AUS/P-8/06). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.