Protein nanocapsules containing doxorubicin as a pH-responsive delivery system

Abstract

The E2 component of pyruvate dehydrogenase is engineered to form a caged, hollow dodecahedral protein assembly, and the feasibility of this scaffold to be used as a drug delivery system is examined by introducing cysteines to the internal cavity (D381C). The fluorescent dye Alexa Fluor 532 (AF532M) and the antitumor drug doxorubicin are coupled to this internal cavity through maleimides on the guest molecules. The viruslike particle's structure and stability remain intact after binding of the molecules within the interior of the nanocapsule. The pH-dependent hydrolysis of a hydrazone linkage to doxorubicin allows 90% drug release from the D381C scaffold within 72 h at pH 5.0. Fluorescence microscopy of MDA-MB-231 breast cancer cells indicates significant uptake of the D381C scaffold incorporating AF532M and doxorubicin, and suggests internalization of the nanoparticles through endocytosis. It is observed that the protein scaffold does not induce cell death, but doxorubicin encapsulated in D381C is indeed cytotoxic, yielding an IC(50) of 1.3 ± 0.3 μM. While the majority of particulate-based drug delivery strategies encapsulates drugs within polymeric nanoparticles, these results show the potential for using macromolecular protein assemblies. This approach yields a promising new opportunity for designing highly defined nanomaterials for therapeutic delivery.

Figure 1

A. Chemical structure of DOXO-EMCH. B. Quaternary protein structure of the E2-D381C nanoparticle, comprising 60 subunits, viewed at the five-fold axis of symmetry. The Protein Data Bank crystallographic file (1b5s) is displayed using PyMOL (DeLano, 2002), and the doxorubicin conjugation sites (amino acid 381) are highlighted in black.

Figure 2

The mostability and structural characterization of protein scaffolds encapsulating doxorubicin (D381C-DOX). A. Far-UV circular dichroism thermostability scan at 222 nm revealed T_m to be 90.5°C. B. Transmission electron micrograph of D381C-DOX. Samples were stained with 2% uranyl acetate. Scale bar is 50 nm.

Figure 3

In vitro release of doxorubicin from D381C-DOX at pH 7.4 and pH 5.0.

Figure 4

MDA-MB-231 cells incubated with (top) free AF532M and (bottom) protein nanoparticle-conjugated AF532M (D381C-AF532M) at different times. Figures are overlays of phase contrast and fluorescence images. Scale bar is 50 μm.

Figure 5

MDA-MB-231 cells incubated with (top) free doxorubicin and (bottom) protein nanoparticle-conjugated doxorubicin (D381C-DOX) at different doxorubicin concentrations for 72 hrs. Figures are overlays of phase contrast and fluorescence images. Scale bar is 50 μm.

Figure 6

Confocal laser scanning microscopy images of MDA-MB-231 cells treated with (top) free doxorubicin and (bottom) protein nanoparticle-conjugated doxorubicin (D381C-DOX) at 3 μM doxorubicin-equivalent concentration. (A) Doxorubicin is red, (B) cell nuclei are blue, and (C) overlay of the two images are presented. Scale bar is 50 μm.

Figure 7

Dose response curves of MDA-MB-231 cells incubated with free doxorubicin and protein nanoparticle-conjugated doxorubicin (D381C-DOX) for 72 hrs. IC₅₀ values are 0.6 ± 0.01 μM for doxorubicin and 1.3 ± 0.3 μM for D381C-DOX.