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We identify specific acylphosphatase (AcP) residues that interact with silica nanoparticles (SNPs) using a combined NMR spectroscopy and proteomics-mass spectrometry approach. AcP associated with 4- and 15-nm diameter SNPs through a common and specific interaction surface formed by amino acids from the two α-helices of the protein. Greater retention of native protein structure was obtained on 4-nm SNPs than on 15-nm particles, presumably due to greater surface curvature-induced protein stabilization with the smaller SNPs. These results demonstrate that proteins may undergo specific and size-dependent orientation on nanoparticle surfaces. Our approach can be broadly applied to various protein-material systems to help understand in much greater detail the protein-nanomaterial interface; it would also encourage better modeling, and thus prediction and design, of the behavior of functional proteins adsorbed onto different surfaces.

Virus-like particles (VLPs) are biological nanoparticles identical to the natural virions, but without genetic material. VLPs are suitable for the analysis of viral infection mechanisms, vaccine production, tissue-specific drug delivery, and as biological nanomaterials. Human parvovirus B19 (B19) infects humans; therefore VLPs derived from this virus have enormous potential in medicine and diagnostics. Current production of self-assembled VLPs derived from B19 is typically carried out in eukaryotic expression systems. However many applications of VLPs require access to its internal core. Consequently, the processes of disassembly and further reassembly of VLPs are critical both for purification of viral proteins, and for encapsulation purposes. Herein we report the in vitro self-assembly of B19 VLPs derived from the recombinant VP2 protein expressed in Escherichia coli and the effects of pH and ionic strength on the assembly process. Our results demonstrate that VP2 is able to form VLPs completely in vitro. At neutral pH, homogeneous VLPs assemble, while at acidic and basic pHs, with low ionic strength, the major assemblies are small intermediates. The in vitro self-assembled VLPs are highly stable at 37°C, and a significant fraction of particles remain assembled after 30min at 80°C., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Many biomedical applications of gold nanoparticles (NPs) rely on proteins that are covalently attached or adsorbed on the NP surface. The biological functionality of the protein-NP conjugate depends on the protein's ability to interact with target molecules, which is affected by NP characteristics such as size, curvature, aspect ratio, morphology, crystal structure, and surface chemistry. In the present study, the effect of gold nanoparticle morphology on the structure and function of adsorbed enzymes, lysozyme (Lyz) and α-chymotrypsin (ChT), has been investigated. Gold nanospheres (AuNS) were synthesized with diameters 10.6 ± 1 nm, and gold nanorods (AuNR) were synthesized with dimensions of (10.3 ± 2) × (36.4 ± 9) nm. Under saturating conditions, proteins adsorb with a higher surface density on AuNR when compared to AuNS. In the case of Lyz, adsorption on AuNS and AuNR resulted in a 10% and 15% loss of secondary structure, respectively, leading to conjugate aggregation and greatly reduced enzymatic activity. ChT retained most of its secondary structure and activity on AuNS and AuNR at low surface coverages; however, as protein loading approached monolayer conditions on AuNR, a 40% loss in secondary structure and 86% loss of activity was observed. Subsequent adsorption of ChT in multilayers on the AuNR surface allowed the conjugates to recover activity and remain stable. It is clear that AuNP morphology does affect adsorbed protein structure; a better understanding of these differences will be essential to engineer fully functional nanobioconjugates., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Intracellular delivery of specific proteins and peptides may be used to influence signaling pathways and manipulate cell function, including stem cell fate. Herein, we describe the delivery of proteins attached to hydrophobically modified 15-nm silica nanoparticles to manipulate specifically targeted cell signaling proteins. We designed a chimeric protein, GFP-FRATtide, wherein GFP acts as a biomarker for fluorescence detection, and FRATtide binds to and blocks the active site of glycogen synthase kinase-3β (GSK-3β) - a protein kinase involved in Wnt signaling. The SiNP-chimeric protein conjugates were efficiently delivered to the cytosol of human embryonic kidney cells and rat neural stem cells, presumably via endocytosis. This uptake impacted the Wnt signaling cascade, resulting in an elevation of β-catenin levels due to GSK-3β inhibition. Accumulation of β-catenin led to increased transcription of Wnt target genes, such as c-MYC, which instruct the cell to actively proliferate and remain in an undifferentiated state. The results presented here suggest that functional proteins can be delivered intracellularly in vitro using nanoparticles and used to target key signaling proteins and regulate cell signaling pathways. This ability is critical for the design of in vitro screens for gain/loss of pathway function, and may also prove to be useful for in vivo delivery applications., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Three-dimensional (3D) cellular assays closely mimic the in vivo milieu, providing a rapid, inexpensive system for screening drug candidates for toxicity or efficacy in the early stages of drug discovery. However, 3D culture systems may suffer from mass transfer limitations, particularly in delivery of large polypeptide or nucleic acid compounds. Nucleic acids (e.g., genes, silencing RNA) are of particular interest both as potential therapeutics and due to a desire to modulate the gene-expression patterns of cells exposed to small-molecule pharmacological agents. In the present study, polyethylenimine (PEI)-coated superparamagnetic nanoparticles (SPMNs) were designed to deliver interfering RNA and green fluorescent protein (GFP) plasmids through a collagen-gel matrix into 3D cell cultures driven by an external magnetic field. The highest transfection efficiency achieved was 64% for siRNA and 77% for GFP plasmids. Delivery of an shRNA plasmid against GFP by PEI-coated SPMNs silenced the GFP expression with 82% efficiency. We further demonstrated that this delivery approach could be used for screening interfering RNA constructs for therapeutic or toxic effects for cells grown in 3D cultures. Four known toxic shRNA plasmids were delivered by PEI-coated SPMNs into 3D cell cultures, and significant toxicities (41-51% cell death) were obtained.

Despite recent advances in nanomaterial-based delivery systems, their applicability as carriers of cargo, especially proteins for targeting cellular components and manipulating cell function, is not well-understood. Herein, we demonstrate the ability of hydrophobic silica nanoparticles to deliver proteins, including enzymes and antibodies, to a diverse set of mammalian cells, including human cancer cells and rat stem cells, while preserving the activity of the biomolecule post-delivery. Specifically, we have explored the delivery and cytosolic activity of hydrophobically functionalized silica nanoparticle-protein conjugates in a human breast cancer cell line (MCF-7) and rat neural stem cells (NSCs) and elucidated the mechanism of cytosolic transport. Importantly, the proteins were delivered to the cytosol without extended entrapment in the endosomes, which facilitated the retention of biological activity of the delivered proteins. As a result, delivery of ribonuclease A (RNase A) and the antibody to phospho-Akt (pAkt) resulted in the initiation of cell death. Delivery of control protein conjugates (e.g., those containing green fluorescent protein or goat antirabbit IgG) resulted in minimal cell death, indicating that the carrier-mediated toxicity was low. The results presented here provide insight into the design of nanomaterials as protein carriers that enable control of cell function.

The structure, thermodynamic and kinetic stability, and activity of cytochrome c (cyt c) on silica nanoparticles (SNPs) of different sizes have been studied. Adsorption of cyt c onto larger SNPs results in both greater disruption of the cyt c global structure and more significant changes of the local heme microenvironment than upon adsorption onto smaller SNPs. The disruption of the heme microenvironment leads to a more solvent-accessible protein active site, as suggested by Soret circular dichroism spectroscopy and through an increase in peroxidase activity as a function of increased SNP size. Similarly, the stability of cyt c decreases more dramatically upon adsorption onto larger SNPs. These results are consistent with changes in protein-nanoparticle interactions that depend on the size or surface curvature of the supporting nanostructure. This study provides further fundamental insights into the effects of nanoscale surfaces on adsorbed protein structure and function.

This paper reports on the unfolding behavior of ribonuclease A (RNase A) on silica nanoparticle surfaces and quantitively demonstrates that nanoscale size and surface curvature play key roles in influencing the stability of adsorbed proteins. Urea denaturation analyses showed that the thermodynamic stability of RNase A decreased upon adsorption onto the nanoparticles, with greater decrease on larger nanoparticles. The stability changes of RNase A correlate well with the changes in the protein-nanoparticle interactions, which increase as the surface contact area and surface charge interaction increases. This study, therefore, provides fundamental information on the effect of nanoscale surfaces on protein structure and function.

We have discovered that the highly curved surface of C60 fullerenes enhances enzyme stability in strongly denaturing environments to a greater extent than flat supports. The half-life of a model enzyme, soybean peroxidase, adsorbed onto fullerenes at 95 degrees C was 117 min, ca. 2.5-fold higher than that of the enzyme adsorbed onto graphite flakes and ca. 13-fold higher than that of the native enzyme. Furthermore, this phenomenon is not unique to fullerenes, but can also be extended to other nanoscale supports including silica and gold nanoparticles. The enhanced stability was exploited in the preparation of highly active and stable polymer-nanocomposite films. The ability to enhance protein stability by interfacing them with nanomaterials may impact numerous fields ranging from the design of diagnostics, sensors, and nanocomposites to drug delivery.

Recent reports have shown that intracellular, (super)paramagnetic ferritin nanoparticles can gate TRPV1, a non-selective cation channel, in a magnetic field. Here, we report the effects of differing field strength and frequency as well as chemical inhibitors on channel gating using a Ca2+-sensitive promoter to express a secreted embryonic alkaline phosphatase (SEAP) reporter. Exposure of TRPV1-ferritin-expressing HEK-293T cells at 30 °C to an alternating magnetic field of 501 kHz and 27.1 mT significantly increased SEAP secretion by ~ 82% relative to control cells, with lesser effects at other field strengths and frequencies. Between 30–32 °C, SEAP production was strongly potentiated 3.3-fold by the addition of the TRPV1 agonist capsaicin. This potentiation was eliminated by the competitive antagonist AMG-21629, the NADPH oxidase assembly inhibitor apocynin, and the reactive oxygen species (ROS) scavenger N-acetylcysteine, suggesting that ROS contributes to magnetogenetic TRPV1 activation. These results provide a rational basis to address the heretofore unknown mechanism of magnetogenetics. [ABSTRACT FROM AUTHOR]

A correction to the article "Remote regulation of glucose homeostasis in mice using genetically encoded nanoparticles," that appeared in the 2015 issue is presented.

For the encapsulation of biomolecules in inorganic materials, we have developed a unique enzyme-silicate conjugate material that consists of a self-assembled molecularly thin silicate layer on the surface of each individual enzyme molecule. The enzyme-silicate conjugate materials, called single enzyme caged nanoparticles (SECNs), were synthesized via the silica polymerization on the surface of enzyme molecule after solubilizing each enzyme molecule in hexane by using a tiny amount of surfactant, called “ion-pairing”. SECNs possess near native enzyme activity in aqueous media with minimal substrate diffusional limitations, and are highly stable under the protection of silicate network cage. Due to their nearly molecular size, SECNs can also be adsorbed into mesoporous silica materials to yield robust and easily-recyclable enzymatic systems that can be used in a number of potential biocatalytic applications such as diagnostics, biosensors, biotransformations, biofuel production, bioremediation and CO 2 capture. [ABSTRACT FROM AUTHOR]

The article discusses a study in which researchers combined nanotechnology and bioengineering to demonstrate the effectiveness of nanoparticles in regulating plasma glucose in mice. They decorated a modified temperature-sensitive channel, called TRPV1, with antibody-coated iron oxide nanoparticles that were heated in a low-frequency magnetic field. Results showed that when local temperature rose, TRPV1 gated calcium to stimulate synthesis and release bioengineered insulin driven by a Ca2+-sensitive promoter. The team's study of tumor xenografts expressing the bioengineered insulin gene showed that exposure to radio waves stimulated insulin release from the tumors and lowered blood glucose in mice.

Using digital microfluidics, recombinant enzyme technology, and magnetic nanoparticles, we have created a functional prototype of an artificial Golgi organelle. Analogous to the natural Golgi, which is responsible for the enzymatic modification of glycosaminoglycans immobilized on proteins, this artificial Golgi enzymatically modifies glycosaminoglycans, specifically heparan sulfate (HS) chains immobilized onto magnetic nanoparticles. Sulfo groups were transferred from adenosine 3'-phosphate 5'-phosphosulfate to the 3-hydroxyl group of the D-glucosamine residue in an immobilized HS chain using D-glucosaminyl 3-O-sulfotransferase. After modification, the nanoparticles with immobilized HS exhibited increased affinity for fluorescently labeled antithrombin Ill as detected by confocal microscopy. Since the biosynthesis of HS involves an array of specialized glycosyl transferases, epimerase, and sulfotransferases, this approach should mimic the synthesis of HS in vivo. Furthermore, our method demonstrates the feasibility of investigating the effects of multienzyme systems on the structure of final glycan products for HS-based glycomic studies. [ABSTRACT FROM AUTHOR]