Disposable biosensors based on metal nanoparticles.

The coronavirus disease2019 (COVID-19) pandemic has highlighted the need for disposable biosensors that can detect viruses in infected patients quickly due to fast response and also at a low cost.The present review provides an overview of the applications of disposable biosensors based on metal nanoparticles in enzymatic and non-enzymatic sensors with special reference to glucose and H2O2, immunosensors as well as genosensors (DNA biosensors in which the recognized event consists of the hybridization reaction)for point-of-care diagnostics. The disposable biosensors for COVID19 have also been discussed.

A comparison between electrospinning and rotary-jet spinning to produce PCL fibers with low bacteria colonization.

One of the important components in tissue engineering is material structure, providing a model for fixing and the development of cells and tissues, which allows for the transport of nutrients and regulatory molecules to and from cells. The community claims the need for new materials with better properties for use in the clinic. Poly (ε-caprolactone) (PCL) is a biodegradable polymer, semi crystalline, with superior mechanical properties and has attracted an increasing interest due to its usefulness in various biomedical applications. Herein, two different methods (electrospinning versus rotary jet spinning) with different concentrations of PCL produced ultra thin-fibers each with particular characteristics, verified and analyzed by morphology, wettability, thermal and cytotoxicity features and for bacteria colonization. Different PCL scaffold morphologies were found to be dependent on the fabrication method used. All PCL scaffolds showed greater mammalian cell interactions. Most impressively, rotary-jet spun fibers showed that a special rough surface decreased bacteria colonization, emphasizing that no nanoparticle or antibiotic was used; maybe this effect is related with physical (scaffold) and/or biological mechanisms. Thus, this study showed that rotary jet spun fibers possess a special topography compared to electrospun fibers to reduce bacteria colonization and present no cytotoxicity when in contact with mammalian cells.

Enhanced blood brain barrier permeability and glioblastoma cell targeting via thermoresponsive lipid nanoparticles.

Thermoresponsive targeting is used to deliver therapeutic agents at hyperthermic conditions (39-45 °C). However, available thermoresponsive drug delivery systems (TDDS), including liposomes, have a complex method of preparation involving toxic solvents and reagents. The objective of this in vitro study was to prepare and characterize thermoresponsive lipid nanoparticles (TLN) for treating glioblastoma, the most aggressive brain tumor whose treatment is limited by a low blood brain barrier (BBB) permeability of drugs. Thermoresponsive lipids were prepared by mixing liquid and solid fatty acids (0.1 : 1 to 2 : 1 ratio) and lipid mixtures exhibiting a solid-liquid phase transition at 39 °C were identified by plotting melting point against liquid contents. TLN were prepared by a hot melt encapsulation method using mono- or double-surfactant systems. TLN showed desirable size (<270 nm), zeta potential (-35 to -50 mV), spherical morphology and stability by FTIR studies. In the drug release studies, paclitaxel release was slow at 37 °C, however, it was released abruptly at 39 °C due to the faster diffusion rate from liquid state nanoparticles. During cytotoxicity studies, the unloaded TLN were non-toxic whereas paclitaxel loaded TLN showed higher cytotoxicity to glioblastoma cells at 39 °C (69% cell viability after one hour) compared to 37 °C (82% cell viability). The TLN showed higher permeability across an in vitro model of BBB at 39 °C due to a deformable liquid state which can squeeze through the tight junctions of the BBB. In conclusion, this study demonstrated that the TLN can be used as a safe and effective alternative to traditional TDDS with higher potential to target glioblastoma cells across the BBB.

Graphene oxide nanoribbons as nanomaterial for bone regeneration: Effects on cytotoxicity, gene expression and bactericidal effect.

Graphene oxide nanoribbons (O-GNR) surges as an interesting nanomaterial for biomedical applications due to feasibility to incorporate functional groups and possible bactericidal properties. Herein, high concentrations of O-GNR were biologically evaluated using human osteoblast cells and gram positive and negative bacteria. Briefly, our goal were to evaluate: (1) synthetic pathway, (2) characterization and (3) effects of O-GNR composition and structural factors as a new approach for biomedical applications. For this, O-GNR were produced combining chemical vapor deposition and oxygen plasma treatment of multiwalled carbon nanotubes. Then, we analyzed the bioactivity, cell viability, osteogenic differentiation, matrix mineralization, mRNA levels of the five genes related direct to bone repair and bactericidal effect of high concentrations of O-GNR (10µgmL-1, 100µgmL-1, 200µgmL-1 and 300µgmL-1). Impressively, O-GNR showed no cytotoxic effects up to a concentration of 100µgmL-1 and no gene expression alteration when used in its dose. We also observed that S. aureus and E. coli bacteria are susceptible to damage when incubated with 100µgmL-1 of O-GNR, showing approximately 50% of bacterial death. We consider that O-GNR displays attractive properties when used at a suitable dose, displaying bactericidal effect and apparently lacking to cause damages in the bone repair process.

Nanostructured poly (lactic acid) electrospun fiber with high loadings of TiO2 nanoparticles: Insights into bactericidal activity and cell viability.

Researchers have been looking for modifying surfaces of polymeric biomaterials approved by FDA to obtain nanofeatures and bactericidal properties. If modified, it would be very interesting because the antibiotic administration could be reduced and, therefore, the bacterial resistance. Here, we report the electrospinning of poly (lactic acid) (PLA) with high loadings of titanium dioxide nanoparticles (TiO2, 1-5wt%) and their bactericidal properties. TiO2 nanoparticles have been recognized for a long time for their antibacterial, low cost and self-cleaning properties. However, their ability to reduce bacteria functions when used in polymers has not been well studied to date. In this context, we aimed here to generate nanostructured PLA electrospun fiber-TiO2 nanoparticle composites for further evaluation of their bactericidal activity and cell viability. TEM and SEM micrographs revealed the successful electrospinning of PLA/TiO2 and the generation of polymer-TiO2 nanostructures. When increasing the TiO2 concentration, we observed a proportional increase in the nanoparticle density along the fiber and surface. The nanostructured PLA/TiO2 nanofibers showed no mammalian cell toxicity and, most importantly, possessed bactericidal activity with higher TiO2 loads. Such results suggest that the present PLA electrospun fiber-TiO2 nanoparticle composites should be further studied for a wide range of biomedical applications.

Inhibition of E. coli and S. aureus with selenium nanoparticles synthesized by pulsed laser ablation in deionized water.

Nosocomial diseases are mainly caused by two common pathogens, Escherichia coli and Staphylococcus aureus, which are becoming more and more resistant to conventional antibiotics. Therefore, it is becoming increasingly necessary to find other alternative treatments than commonly utilized drugs. A promising strategy is to use nanomaterials such as selenium nanoparticles. However, the ability to produce nanoparticles free of any contamination is very challenging, especially for nano-medical applications. This paper reports the successful synthesis of pure selenium nanoparticles by laser ablation in water and determines the minimal concentration required for ~50% inhibition of either E. coli or S. aureus after 24 hours to be at least ~50 ppm. Total inhibition of E. coli and S. aureus is expected to occur at 107±12 and 79±4 ppm, respectively. In this manner, this study reports for the first time an easy synthesis process for creating pure selenium to inhibit bacterial growth.

Anti-infective and osteointegration properties of silicon nitride, poly(ether ether ketone), and titanium implants.

Silicon nitride (Si(3)N(4)) is an industrial ceramic used in spinal fusion and maxillofacial reconstruction. Maximizing bone formation and minimizing bacterial infection are desirable attributes in orthopedic implants designed to adhere to living bone. This study has compared these attributes of Si(3)N(4) implants with implants made from two other orthopedic biomaterials, i.e. poly(ether ether ketone) (PEEK) and titanium (Ti). Dense implants made of Si(3)N(4), PEEK, or Ti were surgically implanted into matching rat calvarial defects. Bacterial infection was induced with an injection of 1×10(4)Staphylococcus epidermidis. Control animals received saline only. On 3, 7, and 14days, and 3months post-surgery four rats per time period and material were killed, and calvariae were examined to quantify new bone formation and the presence or absence of bacteria. Quantitative evaluation of osteointegration to adjacent bone was done by measuring the resistance to implant push-out (n=8 rats each for Ti and PEEK, and n=16 rats for Si(3)N(4)). Three months after surgery in the absence of bacterial injection new bone formation around Si(3)N(4) was ∼69%, compared with 24% and 36% for PEEK and Ti, respectively. In the presence of bacteria new bone formation for Si(3)N(4), Ti, and PEEK was 41%, 26%, and 21%, respectively. Live bacteria were identified around PEEK (88%) and Ti (21%) implants, whereas none were present adjacent to Si(3)N(4). Push-out strength testing demonstrated statistically superior bone growth onto Si(3)N(4) compared with Ti and PEEK. Si(3)N(4) bioceramic implants demonstrated superior new bone formation and resistance to bacterial infection compared with Ti and PEEK.

Enhanced functions of osteoblasts on nanostructured surfaces of carbon and alumina.

It is of the utmost importance to increase the activity of bone cells on the surface of materials used in the design of orthopaedic implants. Increased activity of such cells can promote either integration of these materials into surrounding bone or complete replacement with naturally produced bone if biodegradable materials are used. Osteoblasts are bone-producing cells and, for that reason, are the cells of interest in initial studies of new orthopaedic implants. If these cells are functioning normally, they lay down bone matrix onto both existing bone and prosthetic materials implanted into the body. It is generally accepted that a successful material should enhance osteoblast function, leading to more bone deposition and, consequently, increased strength of the interface between the material and juxtaposed bone. The present study provided the first evidence of greater osteoblast function on carbon and alumina formulations that mimic the nano-dimensional crystal geometry of hydroxyapatite found in bone.

Mechanisms of enhanced osteoblast adhesion on nanophase alumina involve vitronectin.

The role, including concentration, conformation, and bioactivity, of adsorbed vitronectin in enhancing osteoblast adhesion on nanophase alumina was investigated in the present study. Vitronectin adsorbed in a competitive environment in the highest concentration on nanophase alumina compared to conventional alumina. Enhanced adsorption of vitronectin on nanophase alumina was possibly due to decreased adsorption of apolipoprotein A-I and/or increased adsorption of calcium on nanophase alumina. In a novel manner, the present study utilized surface-enhanced Raman scattering (SERS) to determine the conformation of vitronectin adsorbed on nanophase alumina. These results provided the first evidence of increased unfolding of vitronectin adsorbed on nanophase alumina. Increased adsorption of calcium on nanophase alumina may affect the conformation of adsorbed vitronectin specifically to promote unfolding of the macromolecule to expose cell-adhesive epitopes recognized by specific cell-membrane receptors. Results of the present study also provided evidence of dose-dependent inhibition of osteoblast adhesion on nanophase alumina pretreated with vitronectin following preincubation (and thus blocking respective cell-membrane receptors) with either Arginine-Glycine-Aspartic Acid-Serine (RGDS) or Lysine-Arginine-Serine-Arginine (KRSR). These events, namely, enhanced vitronectin adsorption, comformation, and bioactivity, may explain the increased osteoblast adhesion on nanophase alumina.

Enhanced osteoclast-like cell functions on nanophase ceramics.

Synthesis of tartrate-resistant acid phosphatase (TRAP) and formation of resorption pits by osteoclast-like cells, the bone-resorbing cells, on nanophase (that is, material formulations with grain sizes less than 100nm) alumina and hydroxyapatite (HA) were investigated in the present in vitro study. Compared to conventional (that is, grain sizes larger than 100 nm) ceramics, synthesis of TRAP was significantly greater in osteoclast-like cells cultured on nanophase alumina and on nanophase HA after 10 and 13 days, respectively. In addition, compared to conventional ceramics, formation of resorption pits was significantly greater by osteoclast-like cells cultured on nanophase alumina and on nanophase HA after 7, 10, and 13 days, respectively. The present study, therefore, demonstrated, for the first time, enhanced osteoclast-like cell function on ceramic surfaces with nanometer-size surface topography.

Enhanced functions of osteoblasts on nanophase ceramics.

Select functions of osteoblasts (bone-forming cells) on nanophase (materials with grain sizes less than 100 nm) alumina, titania, and hydroxyapatite (HA) were investigated using in vitro cellular models. Compared to conventional ceramics, surface occupancy of osteoblast colonies was significantly less on all nanophase ceramics tested in the present study after 4 and 6 days of culture. Osteoblast proliferation was significantly greater on nanophase alumina, titania, and HA than on conventional formulations of the same ceramic after 3 and 5 days. More importantly, compared to conventional ceramics, synthesis of alkaline phosphatase and deposition of calcium-containing mineral was significantly greater by osteoblasts cultured on nanophase than on conventional ceramics after 21 and 28 days. The results of the present study provided the first evidence of enhanced long-term (on the order of days to weeks) functions of osteoblasts cultured on nanophase ceramics; in this manner, nanophase ceramics clearly represent a unique and promising class of orthopaedic/dental implant formulations with improved osseointegrative properties.

Specific proteins mediate enhanced osteoblast adhesion on nanophase ceramics.

Osteoblast, fibroblast, and endothelial cell adhesion on nanophase (that is, materials with grain sizes less than 100 nm) alumina, titania, and hydroxyapatite (HA) was investigated using in vitro cellular models. Osteoblast adhesion was significantly (p < 0.01) greater after 4 h on nanophase alumina, titania, and HA than it was on conventional formulations of the same ceramics. In contrast, compared to conventional alumina, titania, and HA, after 4 h fibroblast adhesion was significantly (p < 0.01) less on nanophase ceramics. Examination of the underlying mechanism(s) of cell adhesion on nanophase ceramics revealed that these ceramics adsorbed significantly (p < 0.01) greater quantities of vitronectin, which, subsequently, may have contributed to the observed select enhanced adhesion of osteoblasts. Select enhanced osteoblast adhesion was independent of surface chemistry and material phase but was dependent on the surface topography (specifically on grain and pore size) of nanophase ceramics. The capability of synthesizing and processing nanomaterials with tailored (through, for example, specific grain and pore size) structures and topographies to control select subsequent cell functions provides the possibility of designing the novel proactive biomaterials (that is, materials that elicit specific, timely, and desirable responses from surrounding cells and tissues) necessary for improved implant efficacy.

Osteoblast adhesion on nanophase ceramics.

Osteoblast adhesion on nanophase alumina (Al2O3) and titania (TiO2) was investigated in vitro. Osteoblast adhesion to nanophase alumina and titania in the absence of serum from Dulbecco's modified Eagle medium (DMEM) was significantly (P < 0.01) less than osteoblast adhesion to alumina and titania in the presence of serum. In the presence of 10% fetal bovine serum in DMEM osteoblast adhesion on nanophase alumina (23 nm grain size) and titania (32 nm grain size) was significantly (P < 0.05) greater than on conventional alumina (177 nm grain size) and titania (2.12 microm grain size), respectively, after 1, 2, and 4 h. Further investigation of the dependence of osteoblast adhesion on alumina and titania grain size indicated the presence of a critical grain size for osteoblast adhesion between 49 and 67 nm for alumina and 32 and 56 nm for titania. The present study provides evidence of the ability of nanophase alumina and titania to simulate material characteristics (such as surface grain size) of physiological bone that enhance protein interactions (such as adsorption, configuration, bioactivity, etc.) and subsequent osteoblast adhesion.

cDNA cloning and efficient mitochondrial import of pre-mtHSP70 from rat liver.

Members of the 70-kD heat shock protein family have been found in all free-living organisms investigated and in major compartments of eukaryotic cells where they are essential to a wide range of functions, including protein folding and targeting. We have isolated a mitochondrial homolog (mtHSP70) from rat liver using ATP agarose affinity chromatography. Its identity was confirmed on the basis of immunological analysis and Ca(2+)-dependent autophosphorylation. Using protein sequence obtained from the amino termius and nine endo Lys-C peptide fragments, we have employed oligonucleotides to isolate a full-length cDNA clone. The open reading frame encodes a protein of 679 amino acids and calculated M(r) 73,913 daltons. The sequence has a high degree of identity with other members of the HSP70 family, including Escherichia coli DnaK (51%), Saccharomyces cerevisiae SSC1p (65%), the constitutive cytosolic HSP70 from rat, HSC70 (46%), and the rat endoplasmic reticulum isoform, BiP, (49%). The cDNA encodes a precursor protein with a 46-amino-acid signal peptide that is absent from the protein isolated from rat liver. The protein also shows a high degree of identity (98%) with a protein isolated from mouse and human tissues (PBP74, Domanico et al., 1993; mortalin, Wadhwa et al., 1993a; CSA, Michikawa et al., 1993a); however, the intracellular localization of these proteins is uncertain. We show that the precursor of mtHSP70 is efficiently imported into isolated mitochondria from rat liver and processed from 74 kD to the mature 69-kD protein.