Construction of homologous branched oligomer megamolecules based on linker-directed protein assembly.

Utilizing the building blocks of recombinant proteins and synthetic linkers, we have obtained two distinct octameric megamolecules with diverse branched structures. This approach combines principles from both click chemistry and protein engineering technology, enabling the integration of functional domains within highly ordered protein assemblies for biomedical applications.

Synthesis, Characterization, and Simulation of Four-Armed Megamolecules.

This paper describes the synthesis, characterization, and modeling of a series of molecules having four protein domains attached to a central core. The molecules were assembled with the "megamolecule" strategy, wherein enzymes react with their covalent inhibitors that are substituted on a linker. Three linkers were synthesized, where each had four oligo(ethylene glycol)-based arms terminated in a para-nitrophenyl phosphonate group that is a covalent inhibitor for cutinase. This enzyme is a serine hydrolase and reacts efficiently with the phosphonate to give a new ester linkage at the Ser-120 residue in the active site of the enzyme. Negative-stain transmission electron microscopy (TEM) images confirmed the architecture of the four-armed megamolecules. These cutinase tetramers were also characterized by X-ray crystallography, which confirmed the active-site serine-phosphonate linkage by electron-density maps. Molecular dynamics simulations of the tetracutinase megamolecules using three different force field setups were performed and compared with the TEM observations. Using the Amberff99SB-disp + pH7 force field, the two-dimensional projection distances of the megamolecules were found to agree with the measured dimensions from TEM. The study described here, which combines high-resolution characterization with molecular dynamics simulations, will lead to a comprehensive understanding of the molecular structures and dynamics for this new class of molecules.

Multi-responsive and logic controlled release of DNA-gated mesoporous silica vehicles functionalized with intercalators for multiple delivery.

Novel DNA-gated mesoporous silica nanoparticle (MSN) vehicles functionalized with disulfide-linked acridinamine intercalators are constructed for multi-responsive controlled release. The DNA-gated MSN vehicles release cargo encapsulated in the MSN pores under different stimuli, including disulfide reducing agents, elevated temperature, and deoxyribonuclease I (DNase I), for codelivery of drugs and DNA/genes in different forms. Furthermore, the cascade release of encapsulated and intercalative drugs is controlled by AND logic gates in combination of dual stimuli. The ingeniously designed DNA-gated MSN vehicles integrates multiple responses and AND logic gate operations into a single smart nanodevice not only for codelivery of drugs and DNA/genes but also for cascade release of two drugs and has promising biological applications to meet diverse requirements of controlled release.

Kaleidoscope megamolecules synthesis and application using self-assembly technology.

The megamolecules with high ordered structures play an important role in chemical biology and biomedical engineering. Self-assembly, a long-discovered but very appealing technique, could induce many reactions between biomacromolecules and organic linking molecules, such as an enzyme domain and its covalent inhibitors. Enzyme and its small-molecule inhibitors have achieved many successes in medical application, which realize the catalysis process and theranostic function. By employing the protein engineering technology, the building blocks of enzyme fusion protein and small molecule linker can be assembled into a novel architecture with the specified organization and conformation. Molecular level recognition of enzyme domain could provide both covalent reaction sites and structural skeleton for the functional fusion protein. In this review, we will discuss the range of tools available to combine functional domains by using the recombinant protein technology, which can assemble them into precisely specified architectures/valences and develop the kaleidoscope megamolecules for catalytic and medical application.

Appropriate Size of Fe3O4 Nanoparticles for Cancer Therapy by Ferroptosis.

Iron oxide nanoparticles can induce cell death due to the ferroptosis mechanism, showing a great potential for cancer therapy. Here, we synthesized different-sized iron oxide nanoparticles (2-100 nm) to investigate their antitumor effect and toxicity mechanism. It was found that ultrasmall nanoparticles (< ∼5 nm) could accumulate in nucleus and were more efficient in triggering the generation of •OH than larger nanoparticles due to the quicker release of Fe2+, thus exhibiting more remarkable cytotoxicity. Nevertheless, 10 nm iron oxide nanoparticles group displayed the best antitumor effect in vivo. We studied the in vivo and intratumoral biodistribution of the nanoparticles and found that the therapeutic effects were related to both the tumoral accumulation and intratumoral distribution of nanoparticles. This work indicates the appropriate size of Fe3O4 NPs for cancer treatment and illustrates the possible factors that influence the therapeutic effect, suggesting the great potential of iron oxide in clinical application.

Photoactivatable Reaction for Covalent Nanoscale Patterning of Multiple Proteins.

This article describes a photochemical approach for independently patterning multiple proteins to an inert substrate, particularly for studies of cell adhesion. A photoactivatable chloropyrimidine ligand was employed for covalent immobilization of SnapTag fusion proteins on self-assembled monolayers of alkanethiolates on gold. A two-step procedure was used: first, patterned UV illumination of the surface activated protein capture ligands, and second, incubation with a SnapTag fusion protein bound to the surface in illuminated regions. Two different fluorescent proteins were patterned in registry with features of 400 nm in size over a 1 mm2 area. An example is given wherein an anti-carcinoembryonic antigen (anti-CEA) scFv antibody was patterned to direct the selective attachment of a human cancer cell line that express the CEA antigen. This method enables the preparation of surfaces with control over the density and activity of independently patterned proteins.

Combination drug release of smart cyclodextrin-gated mesoporous silica nanovehicles.

An integrated γ-cyclodextrin-gated mesoporous silica delivery system via dual dynamic covalent bonds was constructed with dual drug loading for simultaneous and cascade release in targeted combination drug therapy.

Bifunctional quantum dot-decorated Ag@SiO2 nanostructures for simultaneous immunoassays of surface-enhanced Raman scattering (SERS) and surface-enhanced fluorescence (SEF).

Bifunctional nanostructured ensembles of quantum dot (QD)-decorated Ag@SiO2 nanoparticles embedded with Raman reporters p-aminothiophenol (PATP) were intentionally prepared for simultaneous immunoassays of surface-enhanced Raman scattering (SERS) and surface-enhanced fluorescence (SEF). The maximum SEF intensity was optimized with a silica shell spacer of about 9 nm. The SERS sensitivity was significantly improved due to the large Raman scattering cross-section of the coupling reaction product of PATP, generated on the Ag cores upon irradiation of laser during the SERS measurements. The antibody-immobilized QD-decorated Ag/PATP@SiO2 nanocomposites were used for antigen immunoassays using SERS and SEF with high sensitivity. This progress demonstrates the crucial role of rational design/control of multifunctional nanostructures in biodetection and bioimaging.