General Strategy for Direct Cytosolic Protein Delivery <i>via</i> 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

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

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

Probing the protein–nanoparticle interface: the role of aromatic substitution pattern on affinity

<div><p>A new class of cationic gold nanoparticles (NPs) has been synthesised bearing benzyl moieties featuring –NO₂ and –OMe groups to investigate the regioisomeric control of aromatic NP–protein recognition. In general, NPs bearing electron-withdrawing groups demonstrated higher binding affinities towards green fluorescent protein (GFP) than NPs bearing electron-donating groups. Significantly, a ∼7.5- and ∼4.3-fold increase in binding with GFP was observed for –NO₂ groups in <i>meta-</i>position and <i>para-</i>position, respectively, while <i>ortho</i>-substitution showed binding similar to the unsubstituted ring. These findings demonstrated that the NP–protein interaction can be controlled by tuning the spatial orientation and the relative electronic properties of the aromatic substituents. This improved biomolecular recognition provides opportunities for enhanced biosensing and functional protein delivery to the cells.</p></div

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

CRISPRed Macrophages for Cell-Based Cancer Immunotherapy

We present here an integrated nanotechnology/biology strategy for cancer immunotherapy that uses arginine nanoparticles (ArgNPs) to deliver CRISPR-Cas9 gene editing machinery into cells to generate SIRP-α knockout macrophages. The NP system efficiently codelivers single guide RNA (sgRNA) and Cas9 protein required for editing to knock out the “don’t eat me signal” in macrophages that prevents phagocytosis of cancer cells. Turning off this signal increased the innate phagocytic capabilities of the macrophages by 4-fold. This improved attack and elimination of cancer cells makes this strategy promising for the creation of “weaponized” macrophages for cancer immunotherapy

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

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

Co-Delivery of Protein and Small Molecule Therapeutics Using Nanoparticle-Stabilized Nanocapsules

Combination therapy employing proteins and small molecules provides access to synergistic treatment strategies. Co-delivery of these two payloads is challenging due to the divergent physicochemical properties of small molecule and protein cargos. Nanoparticle-stabilized nanocapsules (NPSCs) are promising for combination treatment strategies since they have the potential to deliver small molecule drugs and proteins simultaneously into the cytosol. In this study, we loaded paclitaxel into the hydrophobic core of the NPSC and self-assembled caspase-3 and nanoparticles on the capsule surface. The resulting combination NPSCs showed higher cytotoxicity than either of the single agent NPSCs, with synergistic action established using combination index values

Array-Based Sensing of Metastatic Cells and Tissues Using Nanoparticle–Fluorescent Protein Conjugates

Rapid and sensitive methods of discriminating between healthy tissue and metastases are critical for predicting disease course and designing therapeutic strategies. We report here the use of an array of gold nanoparticle–green fluorescent protein elements to rapidly detect metastatic cancer cells (in minutes), as well as to discriminate between organ-specific metastases and their corresponding normal tissues through their overall intracellular proteome signatures. Metastases established in a <i>new</i> preclinical non-small-cell lung cancer metastasis model in athymic mice were used to provide a challenging and realistic testbed for clinical cancer diagnosis. Full differentiation between the analyte cell/tissue was achieved with as little as 200 ng of intracellular protein (∼1000 cells) for each nanoparticle, indicating high sensitivity of this sensor array. Notably, the sensor created a distinct fingerprint pattern for the normal and metastatic tumor tissues. Moreover, this array-based approach is unbiased, precluding the requirement of <i>a priori</i> knowledge of the disease biomarkers. Taken together, these studies demonstrate the utility of this sensor for creating fingerprints of cells and tissues in different states and present a generalizable platform for rapid screening amenable to microbiopsy samples