Fluorescent, bioactive protein nanoparticles (prodots) for rapid, improved cellular uptake.

A simple and effective method for synthesizing highly fluorescent, protein-based nanoparticles (Prodots) and their facile uptake into the cytoplasm of cells is described here. Prodots made from bovine serum albumin (nBSA), glucose oxidase (nGO), horseradish peroxidase (nHRP), catalase (nCatalase), and lipase (nLipase) were found to be 15-50 nm wide and have been characterized by gel electrophoresis, transmission electron microscopy (TEM), circular dichroism (CD), fluorescence spectroscopy, dynamic light scattering (DLS), and optical microscopic methods. Data showed that the secondary structure of the protein in Prodots is retained to a significant extent and specific activities of nGO, nHRP, nCatalase, and nLipase were 80%, 70%, 65%, and 50% of their respective unmodified enzyme activities. Calorimetric studies indicated that the denaturation temperatures of nGO and nBSA increased while those of other Prodots remained nearly unchanged, and accelerated storage half-lives of Prodots at 60 °C increased by 4- to 8-fold. Exposure of nGO and nBSA+ nGO to cells indicated rapid uptake within 1-3 h, accompanied by significant blebbing of the plasma membrane, but no uptake has been noted in the absence of nGO. The presence of nGO/glucose in the media facilitated the uptake, and hydrogen peroxide induced membrane permeability could be responsible for this rapid uptake of Prodots. In control studies, FITC alone did not enter the cell, BSA-FITC was not internalized even in the presence of nGO, and there has been no uptake of nBSA-FITC in the absence of nGO. These are the very first examples of very rapid cellular uptake of fluorescent nanoparticles into cells, particularly nanoparticles made from pure proteins. The current approach is a simple and efficient method for the preparation of bioactive, fluorescent protein nanoparticles of controllable size for cellular imaging, and cell uptake is under the control of two separate chemical triggers.

Photophysical studies of an encapsulated neutral guest intercalated into the 2-dimensional space of α-Zr(IV) phosphate.

Our long-range objective of developing surface anchored supramolecular assemblies as artificial light harvesting systems led us to explore the intercalation of guest molecules confined within octaamine hydrochloride (OAm·HCl) in the 2-dimensional galleries of layered inorganic material α-Zr(IV)phosphate (α-ZrP). Photophysical properties of 4,4'-dimethylbenzil, camphorthione, 4,4'-dimethylstilbene, pyrene and coumarin-1 were used to probe the intercalation behavior of OAm capsules within the galleries of α-ZrP. (1)H NMR and emission spectral investigations suggested the inclusion of guests within OAm and also confirmed the stability of host-guest complexes under acidic conditions in water. Stirring guest encapsulated OAm capsule with exfoliated α-ZrP nanosheets resulted in intercalation of the host-guest assembly as a whole in the case of 4,4'-dimethylbenzil, camphorthione, and 4,4'-dimethylstilbene as guests. According to powder X-ray diffraction and emission data these capsules are stable in the galleries of α-ZrP. The fact that the capsules are stable and can be included in α-ZrP nanosheets opens up further opportunities to explore inclusion of two different capsules, one with a donor and the other with an acceptor, and study the energy and electron transfer phenomenon between neutral molecules in α-ZrP galleries.

Nanobio interfaces: charge control of enzyme/inorganic interfaces for advanced biocatalysis.

Specific approaches to the rational design of nanobio interfaces for enzyme and protein binding to nanomaterials are vital for engineering advanced, functional nanobiomaterials for biocatalysis, sensing, and biomedical applications. This feature article presents an overview of our recent discoveries on structural, functional, and mechanistic details of how enzymes interact with inorganic nanomaterials and how they can be controlled in a systematic manner using α-Zr(IV)phosphate (α-ZrP) as a model system. The interactions of a number of enzymes having a wide array of surface charges, sizes, and functional groups are investigated. Interactions are carefully controlled to screen unfavorable repulsions and enhance favorable interactions for high affinity, structure retention, and activity preservation. In specific cases, catalytic activities and substrate selectivities are improved over those of the pristine enzymes, and two examples of high activity near the boiling point of water have been demonstrated. Isothermal titration calorimetric studies indicated that enzyme binding is coupled to ion sequestration or release to or from the nanobio interface, and binding is controlled in a rational manner. We learned that (1) bound enzyme stabilities are improved by lowering the entropy of the denatured state; (2) maximal loadings are obtained by matching charge footprints of the enzyme and the nanomaterial surface; (3) binding affinities are improved by ion sequestration at the nanobio interface; and (4) maximal enzyme structure retention is obtained by biophilizing the nanobio interface with protein glues. The chemical and physical manipulations of the nanobio interface are significant not only for understanding the complex behaviors of enzymes at biological interfaces but also for desiging better functional nanobiomaterials for a wide variety of practical applications.

Metal-enzyme frameworks: role of metal ions in promoting enzyme self-assembly on α-zirconium(IV) phosphate nanoplates.

Previously, an ion-coupled protein binding (ICPB) model was proposed to explain the thermodynamics of protein binding to negatively charged α-Zr(IV) phosphate (α-ZrP). This model is tested here using glucose oxidase (GO) and met-hemoglobin (Hb) and several cations (Zr(IV), Cr(III), Au(III), Al(III), Ca(II), Mg(II), Zn(II), Ni(II), Na(I), and H(I)). The binding constant of GO with α-ZrP was increased ∼380-fold by the addition of either 1 mM Zr(IV) or 1 mM Ca(II), and affinities followed the trend Zr(IV) ≃ Ca(II) > Cr(III) > Mg(II) ≫ H(I) > Na(I). Binding studies could not be conducted with Au(III), Al(III), Zn(II), Cu(II), and Ni(II), as these precipitated both proteins. Zr(IV) increased Hb binding constant to α-ZrP by 43-fold, and affinity enhancements followed the trend Zr(IV) > H(I) > Mg(II) > Na(I) > Ca(II) > Cr(III). Zeta potential studies clearly showed metal ion binding to α-ZrP and affinities followed the trend, Zr(IV) ≫ Cr(III) > Zn(II) > Ni(II) > Mg(II) > Ca(II) > Au(III) > Na(I) > H(I). Electron microscopy showed highly ordered structures of protein/metal/α-ZrP intercalates on micrometer length scales, and protein intercalation was also confirmed by powder X-ray diffraction. Specific activities of GO/Zr(IV)/α-ZrP and Hb/Zr(IV)/α-ZrP ternary complexes were 2.0 × 10(-3) and 6.5 × 10(-4) M(-1) s(-1), respectively. While activities of all GO/cation/α-ZrP samples were comparable, those of Hb/cation/α-ZrP followed the trend Mg(II) > Na(I) > H(I) > Cr(III) > Ca(II) ≃ Zr(IV). Metal ions enhanced protein binding by orders of magnitude, as predicted by the ICPB model, and binding enhancements depended on charge as well as the phosphophilicity/oxophilicity of the cation.

Tuning the activities and structures of enzymes bound to graphene oxide with a protein glue.

Graphene oxide (GO) is being investigated extensively for enzyme and protein binding, but many enzymes bound to GO denature considerably and lose most of their activities. A simple, novel, and efficient approach is described here for improving the structures and activities of enzymes bound to GO such that bound enzymes are nearly as active as those of the corresponding unbound enzymes. Our strategy is to preadsorb highly cationized bovine serum albumin (cBSA) to passivate GO, and cBSA/GO (bGO) served as an excellent platform for enzyme binding. The binding of met-hemoglobin, glucose oxidase, horseradish peroxidase, BSA, catalase, lysozyme, and cytochrome c indicated improved binding, structure retention, and activities. Nearly 100% of native-like structures of all the seven proteins/enzymes were noted at near monolayer formation of cBSA on GO (400% w/w), and all bound enzymes indicated 100% retention of their activities. A facile, benign, simple, and general method has been developed for the biofunctionalization of GO, and this approach of coating with suitable protein glues expands the utility of GO as an advanced biophilic nanomaterial for applications in catalysis, sensing, and biomedicine.

Biofunctionalization of α-zirconium phosphate nanosheets: toward rational control of enzyme loading, affinities, activities and structure retention.

Controlling the properties of enzymes bound to solid surfaces in a rational manner is a grand challenge. Here we show that preadsorption of cationized bovine serum albumin (cBSA) to α-Zr(IV) phosphate (α-ZrP) nanosheets promotes enzyme binding in a predictable manner, and surprisingly, the enzyme binding is linearly proportional to the number of residues present in the enzyme or its volume, providing a powerful, new predictable tool. The cBSA loaded α-ZrP (denoted as bZrP) was tested for the binding of pepsin, glucose oxidase (GOX), tyrosinase, catalase, myoglobin and laccase where the number of residues increased from the lowest value of ∼153 to the highest value of 2024. Loading depended linearly on the number of residues, rather than enzyme charge or its isoelectric point. No such correlation was seen for the binding of these enzymes to α-ZrP nanosheets without the preadsorption of cBSA, under similar conditions of pH and buffer. Enzyme binding to bZrP was supported by centrifugation studies, powder X-ray diffraction and scanning electron microscopy/energy-dispersive X-ray spectroscopy. All the bound enzymes retained their secondary structure and the extent of structure retention depended directly on the amount of cBSA preadsorbed on α-ZrP, prior to enzyme loading. Except for tyrosinase, all enzyme/bZrP biocatalysts retained their enzymatic activities nearly 90-100%, and biofunctionalization enhanced the loading, improved structure retention and supported higher enzymatic activities. This approach of using a chemically modified protein to serve as a glue, with a predictable affinity/loading of the enzymes, could be useful to rationally control enzyme binding for applications in advanced biocatalysis and biomedical applications.