The components in natural self-assembled structured materials form in hierarchical, well-organized layers that have specific functions. Mimicking such complex dynamic assemblies is challenging but researchers have already managed to fabricate ordered discrete structures based on self-assembled DNA, proteins, and proteins with DNA. Although these structures mirror the structural complexity of their biological counterparts their architectures are “fixed” in lattice arrays.

Nanoparticles and proteins are attractive complementary building blocks for making “bricks and mortar” hierarchical architectures, and for incorporating biofunctionality into a structure, explains team leader Vincent Rotello. Until now, such assemblies were either built using self-templating proteins, such as viral capsid proteins, for example, or by exploiting complementary supramolecular interactions between nanomaterials and wild-type proteins. This has meant that these systems were restricted to a relatively narrow range of proteins.

New protein-nanoparticle co-engineering approach

“We have now developed a protein-nanoparticle co-engineering approach that can be used to self-assemble dynamic superstructures,” he says. “These assemblies are organised into multiple hierarchical layers and collapse to form granules, which then further self-organize to generate superstructures that are hundreds of nanometres in size.”

The system developed by Rotello and colleagues is based on a green fluorescent protein (GFP) bearing a genetically incorporated glutamic acid peptide chain (E-tags). This engineered protein self-assembles with 2 nm core gold nanoparticles carrying arginine-terminated ligands (ArgNP) though carboxylate guanidinium interactions to generate ordered nanostructures guided by electrostatic self-assembly.

Electrostatic self-assembly thanks to anionic ‘tails’

“The idea is straightforward in concept,” Rotello tells “We engineer anionic ‘tails’ on the proteins and it is these tails that cause them to assemble electrostatically with the cationic nanoparticles. What is surprising, however, is the hierarchical assembly we observed.”

The superstructures are very easy to generate, he says. “We simply mix the nanoparticles and proteins together in the proper ratio and the assembly takes place in a matter of minutes. They also very nicely deliver the proteins directly to the cytosol – though how they do this is an open question.”

According to the researchers, the size and self-assembled structure of the vehicles also make them promising for both localized and systemic gene editing therapeutics, such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) genes, like CRISPR-associated protein 9 (Cas9), which work by delivering proteins into the cell nucleus. Indeed, they say they have already used their self-assembly strategy to create vehicles containing Cas9 complexed with a guide RNA required for editing.

“These assemblies provide very efficient gene editing in vitro with this system, with in vivo studies planned for the near future,” reveals Rotello.

The work is detailed in ACS Nano DOI: 10.1021/acsnano.6b07258.