Metal ion assisted interface re-engineering of a ferritin nanocage for enhanced biofunctions and cancer therapy

Abstract

The bottom-up self-assembly of protein subunits into supramolecular nanoarchitectures is ubiquitously exploited to recapitulate and expand the features of natural proteins to advance nanoscience in medicine. Various chemical and biological re-engineering approaches are available to render diverse functions in the given proteins. They are, unfortunately, capable of compromising protein integrity and stability after extensive modifications. In this study, we introduce a new protein re-engineering method, metal ion assisted interface re-engineering (MAIR), to serve as a robust and universal strategy to extend the functions of self-assembly proteins by boosting structural features to advance their diverse biomedical applications. In particular, the MAIR strategy was applied to a widely used natural protein, ferritin, as a model protein to coordinate with copper ions in its mutagenic artificial metal binding domain. Structure directed rational protein mutagenesis was carried out at the C2 interface amino acid residues of the ferritin subunit for metal ion coordination site optimization. Copper binding at the artificial binding pocket was highly specific over the other divalent ions present in physiological fluids, and the structurally embedded copper ion in turn strengthened the overall protein integrity and stability. In the presence of isotopic copper-64, the interface re-engineered ferritin worked as a chelator-free molecular nanoprobe with an extraordinarily high specific activity to allow PET imaging of tumors in live animals. We also found that the re-engineered ferritin coordinating with copper ions demonstrates high drug loading capacity of a widely used anti-cancer agent, doxorubicin (DOX), to achieve significant drug retention at the tumor site and enhance tumor regression for improved anti-cancer effects. The MAIR approach, thus, exploited the copper ion to facilitate efficient one-step labeling of mutant ferritin derivatives for simultaneous molecular imaging and drug delivery. The reported interface re-engineering strategy provides an unparalleled opportunity to expand protein biofunctions to serve as a new theranostic agent in cancer research.

Biodegradable Nanoparticles of Polyacrylic Acid–Stabilized Amorphous CaCO3 for Tunable pH‐Responsive Drug Delivery and Enhanced Tumor Inhibition

“…The successful encapsulation of DOX in ACC/PAA NPs can be confirmed by the color change from orange (free DOX) to purple (encapsulated DOX) and more precisely by UV‐Vis spectra (Figure b). The PAA‐DOX and Ca 2+ ‐DOX exhibit similar absorption spectra as free DOX with a characteristic absorption peak at 480 nm, which shifts to 511 nm due to the interactions between anthraquinone groups of DOX and metal ions in basic environment . The Fourier‐transform infrared spectroscopy spectra of ACC‐PAA and DOX@ACC‐PAA NPs (Figure c) show a band at 864 cm −1 and a doublet band at 1413/1459 cm −1 arising from the out‐of‐plane bending and asymmetric stretching vibrations of carbonates, respectively, characteristic for the amorphous structure of typical ACCs .…”

Genetically engineered magnetic nanocages for cancer magneto-catalytic theranostics

“…For example, active targeting ligands, including peptides and antibodies, could be introduced to eMIONs via genetic engineering technology for better tumor accumulation. Apart from this, other robust protein engineering strategies, such as metal ion-assisted interface re-engineering technology 37 , can be used to expand the biofunctions of eMIONs, providing an optimal condition as a nanoreactor for catalysis and magnetism properties. Furthermore, eMIONs have the potential to be a favorable magnetic particle imaging (MPI) agent due to the excellent magnetization and optimal size 38,39 , and the efforts are now underway in our laboratory.…”

Tie2-mediated vascular remodeling by ferritin-based protein C nanoparticles confers antitumor and anti-metastatic activities

“…Ferritin nanoparticles are useful platforms for therapeutic agents. They can be chemically modified to impart functionalities within their interior cavities or on their surfaces because of their unique architecture [17]. Unlike most other proteins, ferritin possesses unique properties, such as high solubility, stability, abundance in blood, and low toxicity, which motivates studies on its use as an ideal nanoplatform with many applications including disease therapy and drug delivery [18].…”

“…Since the activation of PAR-1 signaling by TRAP mimics thrombin activity [15] and results in a cytoprotective response when EPCR is occupied with APCs [15,16], both the PC-Gla and TRAP peptides were attached to the surface of ferritin nanoparticles [11]. Ferritin nanoparticles are useful biocompatible, biodegradable, and nontoxic platforms compared with synthetic polymers [17], and ferritin-binding sites and ferritin internalization have been identified in some tumor cells [18]. Therefore, we used small ferritin as a basic template to create nanoparticles [11].…”