Surface Functionalization of Gold Nanorods Improves Nanostructure Assemblies on Amyloid Fibril Scaffolds.

A hybrid scaffold containing gold nanorods and lysozyme amyloid fibrils has been fabricated, and the effect of surface modification on improving nanostructure assembly on the biological template has been investigated. The nanohybrid system was characterized by monitoring surface plasmon resonance bands, dynamic light scattering spectroscopy, Thioflavin-T assay, and transmission electron microscopy. Surface of gold nanorods (GNRs) was modified with polystyrene sulfonate (PSS), and possible difference in assembly of the pristine and modified nanostructures was compared upon interaction with amyloid fibrils. Analysis of transmission electron microscopy showed that changing the surface charge of GNRs with biocompatible polymer improved electrostatic interactions between the nanostructures and amyloid fibril templates. Analysis of cell viability assays also showed that surface functionalization of GNRs remarkably improved biocompatibility of the nanoscaffold. Results of this study encourage utilization of modification strategies to fabricate a new generation of nanoscaffolds with fruitful applications in regenerative medicine.

Versatile nano-platform for tailored immuno-magnetic carriers.

Custom immuno-magnetic devices are desirable tools for biomedical and biotechnological applications. Herein, surface active maghemite nanoparticles (SAMNs) are proposed as a versatile platform for developing tailored immuno-magnetic nano-carriers by simple wet reactions. Two examples for conjugating native and biotinylated antibodies were presented along with their successful applications in the recognition of specific foodborne pathogens. Nanoparticles were functionalized with rhodamine B isothiocyanate (RITC), leading to a fluorescent nano-conjugate, and used for binding anti-Campylobacter fetus antibodies (SAMN@RITC@Anti-Cf). The microorganism was selectively captured in the presence of two other Campylobacter species (C. jejuni and C. coli), as verified by PCR. Alternatively, SAMNs were modified with avidin, forming a biotin-specific magnetic nano-carrier and used for the immobilization of biotinylated anti-Listeria monocytogenes antibodies (SAMN@avidin@Anti-Lm). This immuno-magnetic carrier was integrated in piezoelectric quartz crystal microbalance (QCM) sensor for the detection of L. monocytogenes in milk, showing a detection limit of 3 bacterial cells. The present work presents a new category of customized immuno-magnetic nano-carriers as a competitive option for suiting specific applications. Graphical abstract ᅟ.

Synthesis and Characterization of a Nano-Polyplex system of GNRs-PDMAEA-pDNA: An Inert Self-Catalyzed Degradable Carrier for Facile Gene Delivery.

Engineering molecules at nano-scale is a promising approach in targeting and curing diseases. In this research, fabricated new hybrid system called nano-polyplex represents an example of the molecular engineering at nano-scale. Polymer of PDMAEAs with four different molecular weights were synthesized using the RAFT method, attached onto the gold nano-rod surface, which modified and produced a safe novel system with an average size less than 100 nm. The hybrid system was characterized by ultra violet-visible spectrophotometer (UV-Vis), dynamic light scattering (DLS), 1H NMR, gel permeation chromatography (GPC), Fourier transform-infrared (FT-IR) spectroscopy, Zeta potential analyzer and transmission electron microscopy (TEM). Features of higher transfection and lower toxicity compared to the previously reported polyplex of PDMAEA, as well as the gold standard PEI, have been shown in all molecular weights and defined N/P ratios (10-200). The ideal physicochemical properties for escaping from the cell barriers, covering the large volume of genetic material (pDNA) and high efficiency of loading polyplexes on GNRs' surface make it an ideal carrier. The results of this effort pave way in designing a new generation of nanoparticle-based delivery systems for nucleic acid therapy and gene editing.

Improving the stability of chondroitinase ABC I via interaction with gold nanorods.

Chondroitinase ABC I (cABC I) cleaves glycosaminoglycan chains which are responsible for most of the inhibition of axon regrowth in spinal cord injury. The application of chondroitinase ABC I (cABC I) in damaged nervous tissue is found to prune glycosaminoglycan chains of proteoglycans and facilitate axon regeneration. However, a limiting factor for such application is the enzyme's instability. In this study, the structure and activity of cABC I have been investigated upon interaction with various concentrations of Gold nanorods. The enzyme preserved its major activity with increase in substrate affinity in the presence of the nanostructures. Analysis of circular dichroism spectropolarimetry data showed that secondary structural content of the enzyme slightly increased. The complex form of the enzyme also showed higher storage stability. Fluorescence studies indicated that enzyme obtained more rigidity in its structure. Taking higher stability of enzyme upon interaction, result of this investigation interaction paves the way for utilizing tiny plasmonic nanostructures for fruitful applications in biomedicine.

Expression, purification, refolding and in vitro recovery of active full length recombinant human gelatinase MMP-9 in Escherichia coli.

Human gelatinase (MMP-9) is a member of matrix metalloproteinases family (MMPs), which has been associated with malignant tumor progression and metastasis by matrix degradation. Herein, active full length recombinant human MMP-9 (amino acid residues 107-707) has been expressed in the form of inclusion bodies in Escherichia coli BL21, using pET21a vector. Solubilization of inclusion bodies was carried out in Tris-HCl buffer with 6 M urea, and refolding was performed using dilution and urea gradient methods. Tris-HCl buffer with 5 mM CaCl2 and 1 µM ZnCl2 at pH 7.8 was used as a refolding buffer. Analysis of the structure by fluorescence and far-UV circular dichroism showed a well-formed structure by urea gradient method. Kinetic parameters in refolding conditions of rhMMP-9 were also analyzed, depicting increase in the enzyme's activity without any aggregation.

Development of a label-free SPR sensor for detection of matrixmetalloproteinase-9 by antibody immobilization on carboxymethyldextran chip.

Surface plasmon resonance (SPR) immunosensor has been widely utilized for monitoring antigen-antibody interactions. The sensor measures changes of refractive index upon binding of analyte molecules to specific ligand immobilized on the sensor chip. This effort reports development of SPR immunosensor for real-time and label-free detection of recombinant human matrix metalloproteinases-9 (MMP-9), which has been associated with malignant tumor progression and metastasis by matrix degradation. MMP-9 was expressed in Escherichia coli BL21 and purified by Ni-NTA agarose column. CMD 50 D was activated by EDC/NHS for immobilization of monoclonal anti-MMP-9. Atomic force microscopy images showed uniform distribution of anti-MMP-9 over the sensor chip. Equilibrium constant (KD), maximum binding capacity (Rmax) and ∆Gb values for interaction of MMP-9 and anti-MMP-9 were 0.4nM, 680 µRIU and -53.51kJ/mol, respectively. Concentration of MMP-9 in saliva samples was determined, with linearity in the range of 10-200ng/mL. The limit of detection was found to be 8pg/mL, being lower than most of the previously reported techniques.

Spectral properties and thermal stability of AS1411 G-quadruplex.

G-quadruplexes are supramolecular structures of G-rich nucleic acid, formed by non-canonical base pairing in the presence of specific environmental inducers. These structures have been vastly considered in diagnostic and therapeutic applications. However, detailed information on structure, optical properties and thermal stability of G-quadruplex potent oligonucleotides is scarce. Herein, optical properties and thermodynamic stability of AS1411 quadruplex is reported for various concentrations of potassium and lead ions. Circular dichroism showed that AS1411 ss-DNA folds into parallel conformation in the presence of metal ions and molecular crowding condition. UV-vis spectroscopy indicated formation of quadruplex and fluorescent spectroscopy revealed intercalation of PicoGreen in its structure, with enhancement of emission intensity upon increment of metal ion concentration. This investigation also proposes high-throughput and reliable analysis of AS1411 quadruplex's thermal stability by real-time PCR technique, which can be further applied for other quadruplex structures.

Structure-function analysis of a new bacterial lipase: effect of local structure reorganization on lipase activity.

Like many other bacterial lipases Pseudomonas aeruginosa lipase has a very strong structure-function relationship. Herein, the effect of structural compactness of lipase has been investigated on its activity. P. aeruginosa HR59 was isolated from burn infection as a new and inexpensive source. Bacterial lipase was extracted from this strain and purified by ion-exchange chromatography. Two divalent metal ions (calcium and magnesium) and three polar organic solvents (methanol, ethanol and iso-propanol) were used to study the structure-function relationship. Results of this study revealed that these additives activated enzyme by reducing the helix content of enzyme. Moreover, thermo stability of the enzyme decreased in the presence of calcium and magnesium ions; whereas it increased upon interaction with polar organic solvents under heat-induced denaturation. Results of this investigation encourage utilization of these activity enhancers for various medical and industrial applications.

Conformation and activity of lysozyme on binding to two types of gold nanorods: a comparative study.

The unique morphology of anisotropic rod-shaped gold nanostructures has offered new prospects for biomedical and biosensing applications. This study investigates the interaction of two types of rod-shaped nanostructures, gold nanorods and gold nanorices with lysozyme as a model protein, comparing the probable structural, activity and kinetic stability alterations. Circular dichroism spectropolarimeter revealed that lysozyme retains high fraction of its native conformation in the presence of both nanostructures, with a slight increase in the helical and beta content. Upon the protein adsorption on both types of nanorods, kinetic studies showed maintenance of enzymatic activity, together with increase in the enzymatic affinity and kinetic stability at high temperature. Comparatively, gold nanorice induced better effect on the activity and stability of enzyme than that of gold nanorod. This study might open new insight into potential applications of gold nanorods as nanocarriers for genes and drugs; provided that the toxicological aspect of cationic surfactant-coated nanostructure is taken into consideration.

Interaction of lysozyme with gold nanorods: conformation and activity investigations.

Gold nanorods, with their unique morphology and optical properties have offered new prospects for biomedical and biosensing applications. Herein, the interaction between gold nanorods and a model protein has been monitored using spectroscopic techniques. The enzyme retains high fraction of its native structure with a slight increase in the helical content at the expense of ß-turns. Kinetic investigations revealed notable increase of enzyme affinity for substrate without significant decrease in the V(max). Emission spectra of tryptophan residues in the presence of chaotropic agent highlighted the maintenance of internal quenching due to the induced compactness. Comparison of the gold nanorod treated lysozyme with free enzyme revealed higher thermodynamic stability under denaturing condition. Results from this study encourage the possibility of utilizing gold nanorods as promising nanocarrier candidates for a new generation of drug delivery applications.

Differential scanning calorimetry techniques: applications in biology and nanoscience.

This paper reviews the best-known differential scanning calorimetries (DSCs), such as conventional DSC, microelectromechanical systems-DSC, infrared-heated DSC, modulated-temperature DSC, gas flow-modulated DSC, parallel-nano DSC, pressure perturbation calorimetry, self-reference DSC, and high-performance DSC. Also, we describe here the most extensive applications of DSC in biology and nanoscience.