Moumita Ray

Direct Cytosolic Delivery of CRISPR/Cas9-Ribonucleoprotein for Efficient Gene Editing

Genome editing through the delivery of CRISPR/Cas9-ribonucleoprotein (Cas9-RNP) reduces unwanted gene targeting and avoids integrational mutagenesis that can occur through gene delivery strategies. Direct and efficient delivery of Cas9-RNP into the cytosol followed by translocation to the nucleus remains a challenge. Here, we report a remarkably highly efficient (∼90%) direct cytoplasmic/nuclear delivery of Cas9 protein complexed with a guide RNA (sgRNA) through the coengineering of Cas9 protein and carrier nanoparticles. This construct provides effective (∼30%) gene editing efficiency and opens up opportunities in studying genome dynamics.

Quantitative Tracking of Protein Trafficking to the Nucleus Using Cytosolic Protein Delivery by Nanoparticle-Stabilized Nanocapsules

We describe a method for quantitative monitoring of subcellular protein trafficking using nanoparticle-stabilized nanocapsules for protein delivery. This method provides rapid delivery of the protein into the cytosol, eliminating complications from protein homeostasis processes found with cellularly expressed proteins. After delivery, nuclear protein trafficking was followed by real time microscopic imaging. Quantitative analyses of the accumulation percentage and the import dynamics of the nuclear protein trafficking, demonstrate the utility of this method for studying intracellular trafficking systems.

In Vivo Delivery of CRISPR/Cas9 for Therapeutic Gene Editing: Progress and Challenges

The successful use of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-based gene editing for therapeutics requires efficient in vivo delivery of the CRISPR components. There are, however, major challenges on the delivery front. In this Topical Review, we will highlight recent developments in CRISPR delivery, and we will present hurdles that still need to be overcome to achieve effective in vivo editing.

Engineering the nanoparticle-protein interface for cancer therapeutics.

Intracellular delivery of functional proteins using nanoparticles can be a game-changing approach for cancer therapy. However, cytosolic release of functional protein is still a major challenge. In addition, formation of protein corona on the surface of the nanoparticles can also alter the behavior of the nanoparticles. Here, we will review recent strategies for protein delivery into the cell. Finally we will discuss the issue of protein corona formation in light of nanoparticle-protein interactions.

Cytosolic delivery of large proteins using nanoparticle-stabilized nanocapsules

We report an effective intracellular delivery strategy for proteins of high molecular weight using AuNP stabilized capsules. This strategy provides direct delivery to the cytosol, avoiding endosomal entrapment.

Programmed Self-Assembly of Hierarchical Nanostructures through Protein–Nanoparticle Coengineering

Hierarchical organization of macromolecules through self-assembly is a prominent feature in biological systems. Synthetic fabrication of such structures provides materials with emergent functions. Here, we report the fabrication of self-assembled superstructures through coengineering of recombinant proteins and nanoparticles. These structures feature a highly sophisticated level of multilayered hierarchical organization of the components: individual proteins and nanoparticles coassemble to form discrete assemblies that collapse to form granules, which then further self-organize to generate superstructures with sizes of hundreds of nanometers. The components within these superstructures are dynamic and spatially reorganize in response to environmental influences. The precise control over the molecular organization of building blocks imparted by this protein-nanoparticle coengineering strategy provides a method for creating hierarchical hybrid materials.

General Strategy for Direct Cytosolic Protein Delivery via Protein–Nanoparticle Co-engineering

Endosomal entrapment is a key hurdle for most intracellular protein-based therapeutic strategies. We report a general strategy for efficient delivery of proteins to the cytosol through co-engineering of proteins and nanoparticle vehicles. The proteins feature an oligo(glutamate) sequence (E-tag) that binds arginine-functionalized gold nanoparticles, generating hierarchical spherical nanoassemblies. These assemblies fuse with cell membranes, releasing the E-tagged protein directly into the cytosol. Five different proteins with diverse charges, sizes, and functions were effectively delivered into cells, demonstrating the generality of our method. Significantly, the engineered proteins retained activity after cytosolic delivery, as demonstrated through the delivery of active Cre recombinase, and granzyme A to kill cancer cells.

Cellular imaging of endosome entrapped small gold nanoparticles.

Graphical abstract

Intracellular delivery of proteins by nanocarriers

Intracellular delivery of proteins is potentially a game-changing approach for therapeutics. However, for most applications, the protein needs to access the cytosol to be effective. A wide variety of strategies have been developed for protein delivery, however access of delivered protein to the cytosol without acute cytotoxicity remains a critical issue. In this review we discuss recent trends in protein delivery using nanocarriers, focusing on the ability of these strategies to deliver protein into the cytosol.

Chemically Engineered Nanoparticle-Protein Interface for Real-Time Cellular Oxidative Stress Monitoring.

A co-engineered nanoparticle/protein peroxide detector is created. This system features a gold nanoparticle functionalized with a galactose headgroup (AuNP-Gal) that reacts covalently with a boronate-modified green fluorescent protein (PB-GFP). Boronate acid-saccharide complexation between PB-GFP and AuNP-Gal affords a highly stable assembly. This complex is disrupted by peroxide, allowing quantitative and selective monitoring of hydrogen peroxide production in real time.