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

Influence of Hierarchical Interfacial Assembly on Lipase Stability and Performance in Deep Eutectic Solvent

Hierarchical systems that integrate nano- and macroscale structural elements can offer enhanced enzyme stability over traditional immobilization methods. Microparticles were synthesized using interfacial assembly of lipase B from <i>Candida antarctica</i> with (CLMP-N) and without (CLMP) nanoparticles around a cross-linked polymeric core, to characterize the influence of the hierarchical assembly on lipase stability in extreme environments. Kinetic analysis revealed that the turnover rate (<i>k</i>_cat) significantly increased after immobilization. The macrostructure stabilized lipase at neutral and basic pH values, while the nanoparticles influenced stability under acidic pH conditions. Performance of CLMPs was demonstrated by production of sugar ester surfactants in a greener, deep eutectic solvent system (choline chloride and urea). Turnover rate (<i>k</i>_cat) and catalytic efficiency (<i>k</i>_cat/<i>K</i>_m) of the CLMPs decreased following solvent exposure but retained over 60% and 20% activity after 48 h storage at 50 and 60 °C, respectively. CLMP and CLMP-N outperformed the commercially available lipase per unit protein in the production of sugar esters. Improving enzyme performance in greener solvent systems via hierarchical assembly can improve processing efficiency and sustainability for the production of value-added agricultural products

Charge-Switchable Nanozymes for Bioorthogonal Imaging of Biofilm-Associated Infections

Early detection of biofilms is crucial for limiting infection-based damage. Imaging these biofilms is challenging: conventional imaging agents are unable to penetrate the dense matrix of the biofilm, and many imaging agents are susceptible to false positive/negative responses due to phenotypical mutations of the constituent microbes. We report the creation of pH-responsive nanoparticles with embedded transition metal catalysts (nanozymes) that effectively target the acidic microenvironment of biofilms. These pH-switchable nanozymes generate imaging agents through bioorthogonal activation of profluorophores inside biofilms. The specificity of these nanozymes for imaging biofilms in complex biosystems was demonstrated using coculture experiments

Tuning DNA Condensation with Zwitterionic Polyamidoamine (zPAMAM) Dendrimers

Cationic dendrimers are promising vectors for nonviral gene therapies due to their well-defined size and chemistry. We have synthesized a series of succinylated fourth generation (G4) PAMAM dendrimers to control the DNA packaging in dendriplexes, allowing us to probe the role of charge on DNA packaging. The self-assembly of DNA induced by these zwitterionic PAMAM (zPAMAM) was investigated using small-angle X-ray scattering (SAXS). We demonstrate that changing the degree of modification in zPAMAM–DNA significantly alters the packing density of the resulting dendriplexes. Salt sensitivities and pH dependence on the inter-DNA spacing were also examined. The swelling and stability to salt are reduced with increasing degree of PAMAM modification. Lowering the pH leads to significantly tighter hexagonal DNA packaging. In combination, these results show zPAMAM is an effective means to modulate nucleic acid packaging in a deterministic manner

Development of Engineered Bacteriophages for Escherichia coli Detection and High-Throughput Antibiotic Resistance Determination

T7 bacteriophages (phages) have been genetically engineered to carry the <i>lacZ</i> operon, enabling the overexpression of beta-galactosidase (β-gal) during phage infection and allowing for the enhanced colorimetric detection of Escherichia coli (E. coli). Following the phage infection of E. coli, the enzymatic activity of the released β-gal was monitored using a colorimetric substrate. Compared with a control T7 phage, our T7_<i>lacZ</i> phage generated significantly higher levels of β-gal expression following phage infection, enabling a lower limit of detection for E. coli cells. Using this engineered T7_<i>lacZ</i> phage, we were able to detect E. coli cells at 10 CFU·mL^–1 within 7 h. Furthermore, we demonstrated the potential for phage-based sensing of bacteria antibiotic resistance profiling using our T7_<i>lacZ</i> phage, and subsequent β-gal expression to detect antibiotic resistant profile of E. coli strains

Zwitterionic Ligands Bound to Cdse/Zns Quantum Dots Prevent Adhesion to Mammalian Cells

<div><p></p><p>Zwitterionic materials are useful tools in material science and biology as they provide high water solubility while preventing nonspecific interactions. Quantum dots (QDs) functionalized with zwitterionic and quaternary ammonium ligands were synthesized to investigate their interactions with the outer membrane of HeLa cells. Quaternary ammonium functionalized quantum dots adhered strongly to the cell surface while zwitterionic QDs had no cell adhesion. These results demonstrate that future noninteracting nanoparticles based on this design are possible.</p></div

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

Toward Virus-Like Surface Plasmon Strain Sensors

The strong configuration dependence of collective surface plasmon resonances in an array of metal nanoparticles provides an opportunity to develop a bioinspired tool for sensing mechanical deformations in soft matter at the nanoscale. We study the feasibility of a strain sensor based on an icosahedral array of nanoparticles encapsulated by a virus capsid. When the system undergoes deformation, the optical scattering cross-section spectra as well as the induced electric field profile change. By numerical simulations, we examine how these changes depend on the symmetry and extent of the deformation and on both the propagation direction and polarization of the incident radiation. Such a sensor could prove useful in studies of the mechanisms of nanoparticle or virus translocation in the confines of a host cell

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

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