Doxorubicin-conjugated chimeric polypeptide nanoparticles that respond to mild hyperthermia

Abstract

This paper reports the design, physicochemical characterization and in vitro cytotoxicity of a thermally responsive chimeric polypeptide (CP), derived from an elastin-like polypeptide (ELP). The CP self-assembles into ~40 nm diameter nanoparticles upon conjugation of multiple copies of doxorubicin (Dox), and displays a nanoparticle-to-aggregate phase transition between 39 and 42 °C in media, a temperature range suitable for mild hyperthermia of solid tumors. The CP-Dox nanoparticle is stable upon dilution to low micromolar concentrations, and is cytotoxic at both 37 and 42 °C. A thermally responsive nanoparticle formulation of Dox may prove to be broadly useful in hyperthermia targeted chemotherapy of a variety of solid tumors.

Figure 1

Inverse transition temperature (T_t) of a series of: (A) CPs and (B) CP-Dox nanoparticles as a function of CP concentration in PBS. The dashed line represents the best fit.

Figure 2

(A) Dynamic light scattering results for the CP4 unimer without Dox conjugation (blue) and the CP-Dox nanoparticle (red) at 25 µM CP concentration in PBS at 25 °C. (B) Turbidity profiles of CP4 unimer without Dox conjugation (blue) and the CP-Dox nanoparticle (red) at 25 µM CP concentration in PBS. The shaded region indicates the hyperthermia window (39 – 42 °C) of interest for in vivo thermal targeting. (C) The CP4-Dox nanoparticle (red) shows virtually no dependence of its T_t on concentration and hence remains within the hyperthermia window (black lines) over a wide concentration range whereas the CP4 unimer (blue) and a CP unimer commonly used in previous hyperthermia studies (ELP5: green; X=A₂G₃V₅; data from [17]) show a strong concentration dependence and hence are thermally responsive over a narrow range of concentration. (D) Representative relationship between the T_t and the concentration for CP4 unimer (blue line) and the CP-Dox nanoparticle (red line). The inset displays a magnified view of the low concentration regime for the CP4 unimer and CP4-Dox nanoparticle.

Figure 3

In vitro cytotoxicity of CP-Dox nanoparticles. (A) Fluorescence microscopy of C26 cells following 1 h incubation at 37 °C or (B) 42 °C with CP-Dox nanoparticles at 5.6 µM (30 µM Dox equivalents). Colocalization of Dox (red) with Hoechst stain (blue) suggests that the Dox localizes to the nucleus with and without the application of heat. Scale bars indicate 25 µm. (C) The phase transition of CP4-Dox is fully reversible in complete cell media. CP4-Dox transitions within the hyperthermia window (shaded) at 40.2 °C at 25 µM CP. (D) Viability of C26 cells after mild hyperthermia (1 h at 42 °C) and 24 h incubation with Dox at 37 °C (red) or CP-Dox nanoparticles (blue). (E) Viability of C26 cells following a 1 h exposure at 37 °C or (F) 42 °C and a 72 h incubation in Dox (red) or CP-Dox nanoparticles (blue). Concentrations below 0.1 µM likely result in the disassembly of the nanoparticle system.