Single-cell electroporation

Single-cell electroporation (SCEP) is a relatively new technique that has emerged in the last decade or so for single-cell studies. When a large enough electric field is applied to a single cell, transient nano-pores form in the cell membrane allowing molecules to be transported into and out of the cell. Unlike bulk electroporation, in which a homogenous electric field is applied to a suspension of cells, in SCEP an electric field is created locally near a single cell. Today, single-cell-level studies are at the frontier of biochemical research, and SCEP is a promising tool in such studies. In this review, we discuss pore formation based on theoretical and experimental approaches. Current SCEP techniques using microelectrodes, micropipettes, electrolyte-filled capillaries, and microfabricated devices are all thoroughly discussed for adherent and suspended cells. SCEP has been applied in in-vivo and in-vitro studies for delivery of cell-impermeant molecules such as drugs, DNA, and siRNA, and for morphological observations.     

Bio-electrosprays: from bio-analytics to a generic tool for the health sciences

Electrosprays or electrospraying is a process by which an aerosol is generated between two charged electrodes. This aerosol generation methodology has been known for well over a century, and has undergone exploration in aerosol and materials sciences, to many other areas of research and development. In one such exploration, electrosprays were partnered with mass spectrometry for the accurate characterisation of molecules. This technology now widely referred to as electrospray ionisation mass spectrometry (ESI MS) significantly contributes to molecular analysis and cancer biology to name a few. In fact these findings were recognised by the Chemistry Nobel Committee in 2002, and have catapulted electrosprays to many areas of research and development. In this review, the author wishes to introduce and discuss another such recent discovery, where electrosprays have been investigated for directly handling living cells and whole organisms. Over the past few years these electrosprays now referred to as "bio-electrosprays" have undergone rigorous developmental studies both in terms of understanding all the associate physical, chemical and biological sciences for completely assessing their effects, if any on the direct handling of living biological materials. Therefore, the review will bring together all the work that has contributed to fully understanding that bio-electrosprays are an inert technology for directly handling living biological materials, while elucidating some unique features they possess over competing technologies. Hence, demonstrating this approach as a flexible methodology for a wide range of applications spanning bio-analytics, diagnostics to the possible creation of synthetic tissues, for repairing and replacing damaged/ageing tissues, to the targeted and controlled delivery of personalised medicine through experimental and/or medical cells and/or genes. Therefore, elucidating the far reaching ramifications bio-electrosprays have to our health sciences and well-being.     

Unique Possibilities Offered by Radio-Tracers for Speciation Analysis of Trace Elements in Biological Fluids and Tissues

Speciation analysis of trace elements is the identification and/or measurement of the quantities of one or more individual chemical species in a sample. The general procedure starts with the separation of the species followed by the measurement of trace elements in the different fractions. The identification of the species may be done by UV-detection, mass spectrometry or other techniques. For the development of the methodology, and for in-vivo and in-vitro studies radiotracers are ideally suited. In speciation analysis of trace elements in biological fluids and tissues the search goes on for small inorganic and organometallic compounds, and for metal-protein complexes.     

Plant phenolics: extraction, analysis and their antioxidant and anticancer properties

Phenolics are broadly distributed in the plant kingdom and are the most abundant secondary metabolites of plants. Plant polyphenols have drawn increasing attention due to their potent antioxidant properties and their marked effects in the prevention of various oxidative stress associated diseases such as cancer. In the last few years, the identification and development of phenolic compounds or extracts from different plants has become a major area of health- and medical-related research. This review provides an updated and comprehensive overview on phenolic extraction, purification, analysis and quantification as well as their antioxidant properties. Furthermore, the anticancer effects of phenolics in-vitro and in-vivo animal models are viewed, including recent human intervention studies. Finally, possible mechanisms of action involving antioxidant and pro-oxidant activity as well as interference with cellular functions are discussed.     

In-Vivo Analysis and Model-Based Prediction of Tensides’ Influence on Drug Absorption

Depending on their concentrations the surface-active substances, tensides (surfactants) can positively or negatively influence the drug absorption, which is widely used in the design of the dosage forms with controlled release. A problem is that the (in-vivo) rate of absorption cannot be directly measured and for that reason, it is frequently substituted by evaluation of the (in-vitro) dissolution. On other hand, a suitably designed pharmacokinetic model can directly predict virtually all pharmacokinetic quantities including both the rate of absorption and fraction of the dose reaching the blood circulation. The paper presents a new approach to the analysis of the rate of drug absorption and shows its superiority over traditional in-vivo approaches. Both the in-vivo analysis and model-based prediction of the tenside (monolaurin of sucrose) influence on the rate of absorption of the drug (sulfathiazole) after instantaneous per-oral administration to rats are discussed. It was found that 0.001% solution of tenside can increase the rate of absorption by cca 50% and a two-fold increase in absolute bioavailability can be reached. Attention is also devoted to the formal requirements laid on the model’s structure and its identifiability. The systematic design, substantiation and validation of a parsimonious predictive model that confirms in-vivo results are presented. The match between in-vivo observations and model-based predictions is demonstrated. The frequently overlooked metaphysics lying behind the compartmental modelling is briefly explained.     

Real-time cell-impedance sensing assay as an alternative to clonogenic assay in evaluating cancer radiotherapy

Intrinsic radiosensitivity of normal and tumour tissues has been shown to be an independent prognostic factor for patients’ response to radiotherapy. This study compares the real-time cell-impedance sensing (RT-CES) assay with the conventional clonogenic assay in terms of in-vitro radiosensitivity. One objective in this study was to predict in-vivo response to gold nanoparticle (GNP) treatment on the basis of in-vitro RT-CES testing results. Four adenocarcinoma cancer cell lines were tested using both the RT-CES and the clonogenic assays. Cell-survival curves were plotted, and the mean SF2 values obtained by these two different assay methods were compared using ANOVA. Radiation sensitivities obtained in-vitro were then compared with the in-vivo results. On the basis of the measurement of cell colonies, the RT-CES assay has similar radiosensitivity to the clonogenic assay, but significantly shortens the testing time from 14–21 days to only 72 h. Intrinsic GNP enhanced radiation sensitivity using tumour volume (mm³) in vivo is comparable with that using RT-CES cell survival assay in vitro. Furthermore, the RT-CES system provides real-time information regarding the state of cell radiosensitivity that may give useful information towards personalizing radiotherapy. The RT-CES assay enables more reliable and time-efficient results in the evaluation of radiosensitivity.     

An efficient way to functionalize graphene sheets with presynthesized polymer via ATNRC chemistry

Graphene nanosheets offer intriguing electronic, thermal and mechanical properties and are expected to find a variety of applications in high‐performance nanocomposite materials. The great challenge of exfoliating and dispersing pristine graphite or graphene sheets in various solvents or matrices can be achieved by facilely and properly chemical functionalization of the carbon nanosheets. Here we reported an efficient way to functionalize graphene sheets with presynthesized polymer via a combination of atom transfer nitroxide radical coupling chemistry with the grafting‐onto strategy, which enable us to functionalize graphene sheets with well‐defined polymer synthesized via living radical polymerization. A radical scavenger species, 2,2,6,6‐tetramethylpiperidine‐1‐oxyl (TEMPO), was firstly anchored onto ? COOH groups on graphene oxide (GO) to afford TEMPO‐functionalized graphene sheets (GS‐TEMPO), meanwhile, the GO sheets were thermally reduced. Next, GS‐TEMPO reacted with Br‐terminated well‐defined poly(N‐isopropylacrylamide) (PNIPAM) homopolymer, which was presynthesized by SET‐LRP, in the presence of CuBr/N,N,N′,N′,N″‐pentamethyldiethylenetriamine to form PNIPAM‐graphene sheets (GS‐PNIPAM) nanocomposite in which the polymers were covalently linked onto the graphene via the alkoxyamine conjunction points. The PNIPAM‐modified graphene sheets are easily dispersible in organic solvents and water, and a temperature‐induced phase transition was founded in the water suspension of GS‐PNIPAM. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Synthesis,Characterization and Evaluation of Peptide Nanostructures for Biomedical Applications

There is a challenging need for the development of new alternative nanostructures that can allow the coupling and/or encapsulation of therapeutic/diagnostic molecules while reducing their toxicity and improving their circulation and in-vivo targeting. Among the new materials using natural building blocks, peptides have attracted significant interest because of their simple structure, relative chemical and physical stability, diversity of sequences and forms, their easy functionalization with (bio)molecules and the possibility of synthesizing them in large quantities. A number of them have the ability to self-assemble into nanotubes, -spheres, -vesicles or -rods under mild conditions, which opens up new applications in biology and nanomedicine due to their intrinsic biocompatibility and biodegradability as well as their surface chemical reactivity via amino- and carboxyl groups. In order to obtain nanostructures suitable for biomedical applications, the structure, size, shape and surface chemistry of these nanoplatforms must be optimized. These properties depend directly on the nature and sequence of the amino acids that constitute them. It is therefore essential to control the order in which the amino acids are introduced during the synthesis of short peptide chains and to evaluate their in-vitro and in-vivo physico-chemical properties before testing them for biomedical applications. This review therefore focuses on the synthesis, functionalization and characterization of peptide sequences that can self-assemble to form nanostructures. The synthesis in batch or with new continuous flow and microflow techniques will be described and compared in terms of amino acids sequence, purification processes, functionalization or encapsulation of targeting ligands, imaging probes as well as therapeutic molecules. Their chemical and biological characterization will be presented to evaluate their purity, toxicity, biocompatibility and biodistribution, and some therapeutic properties in vitro and in vivo. Finally, their main applications in the biomedical field will be presented so as to highlight their importance and advantages over classical nanostructures.     

Liquid crystals in biology I. Historical,biological and medical aspects

In our present state of knowledge, it is useful to assume that all matter, in the solar galaxy at least, is composed of atoms and subatomic particles which function independently or interact in accordance with the laws of physics to form molecules, coacervates or other aggregates. For practical purposes, these states of matter are recognizable in the three-dimensional terrestrial world as solids, liquids and gases. This differentiation suffices also for molecular studies but, to understand the properties of mobile organic and especially of living matter fundamentally, it is necessary to investigate and conceptualize how immaterial electromagnetic and electrostatic processes produce changes in state, phase and entropy compatible with self-replication, molecular memory and vitality This possibility exists in the properties of the liquid crystal (LC) as a mesophase in thermal and optical phase transitions, i.e. as an enantiomorphic intermediate form of matter which can form complex, self-replicating, ordered structures and macromolecules, easily recognizable in everyday TV visual displays, electronic communication devices and computers. It is suggested that, in prebiotic terrestrial situations, matter possessing these properties of the LC was a precursor in the evolution of living from inanimate matter and, in the lyotropic form, in the processes of life thereafter.     

A close look at fluorescence quenching of organic dyes by tryptophan.

Understanding fluorescence quenching processes of organic dyes by biomolecular compounds is of fundamental importance for in-vitro and in-vivo fluorescence studies. It has been reported that the excited singlet state of some oxazine and rhodamine derivatives is efficiently and almost exclusively quenched by the amino acid tryptophan (Trp) and the DNA base guanine via photoinduced electron transfer (PET). We present a detailed analysis of the quenching interactions between the oxazine dye MR121 and Trp in aqueous buffer. Steady-state and time-resolved fluorescence spectroscopy, together with fluorescence correlation spectroscopy (FCS), reveal three contributing quenching mechanisms: 1) diffusion-limited dynamic quenching with a bimolecular quenching rate constant k(d) of 4.0 x 10(9) s(-1) M(-1), 2) static quenching with a bimolecular association constant K(s) of 61 M(-1), and 3) a sphere-of-action contribution to static quenching described by an exponential factor with a quenching constant lambda of 22 M(-1). The latter two are characterized as nonfluorescent complexes, formed with approximately 30 % efficiency upon encounter, that are stable for tens of nanoseconds. The measured binding energy of 20-30 kJ mol(-1) is consistent with previous estimates from molecular dynamics simulations that proposed stacked complexes due to hydrophobic forces. We further evaluate the influence of glycerol and denaturant (guanidine hydrochloride) on the formation and stability of quenched complexes. Comparative measurements performed with two other dyes, ATTO 655 and Rhodamine 6G show similar results and thus demonstrate the general applicability of utilizing PET between organic dyes and Trp for the study of conformational dynamics of biopolymers on sub-nanometer length and nanosecond time-scales.     

Micro‐Nanostructures of Cellulose‐Collagen for Critical Sized Bone Defect Healing

Bone tissue engineering strategies utilize biodegradable polymeric matrices alone or in combination with cells and factors to provide mechanical support to bone, while promoting cell proliferation, differentiation, and tissue ingrowth. The performance of mechanically competent, micro‐nanostructured polymeric matrices, in combination with bone marrow stromal cells (BMSCs), is evaluated in a critical sized bone defect. Cellulose acetate (CA) is used to fabricate a porous microstructured matrix. Type I collagen is then allowed to self‐assemble on these microstructures to create a natural polymer‐based, micro‐nanostructured matrix (CAc). Poly (lactic‐co‐glycolic acid) matrices with identical microstructures serve as controls. Significantly higher number of implanted host cells are distributed in the natural polymer based micro‐nanostructures with greater bone density and more uniform cell distribution. Additionally, a twofold increase in collagen content is observed with natural polymer based scaffolds. This study establishes the benefits of natural polymer derived micro‐nanostructures in combination with donor derived BMSCs to repair and regenerate critical sized bone defects. Natural polymer based materials with mechanically competent micro‐nanostructures may serve as an alternative material platform for bone regeneration.     

Novel Designed Polyisobutylene-Based Biopolymers: Synthesis,Characterization, and Biological Testing of Amphiphilic Chameleon Networks

After a very brief introduction into the impact of living polymerization on preparative polymer chemistry and a look into the dearth of de novo polymer synthesis research for novel biopolymers, this presentation will focus on a new class of synthetic biopolymers: amphiphilic chame- leon networks, i.e., biocomponent networks comprising random strands of hydrophobic and hydrophilic polymers, for blood contact application and mainly for use as narrow diameter (<4 mm) vascular grafts. First, the precision syntheses of amphiphilic networks will be outlined. Subse-quently the surface and bulk characterization of these novel molecular composites by a battery of physicochemical methods will be highlighted. Finally, representative results of biological in-vitro and in-vivo testing will be summarized. We propose that for bio-or hemocompatibility to arise, it may be necessary to employ “smart” amphiphilic surfaces capable of rapid reversible hydrophobic/hydrophilic reorganization so as to present the most favorable lowest energy surface conformation to the medium. It appears that these surfaces are smooth, flexible, and of very low modulus, and that the bulk of these materials have cocontinuous phase-separated microarchitectures with random microdomains in the 10–100 A diameter range. Contemporary macromolecular engineering can deliver materials exhibiting this combination of characteristics.     

Aerodynamically assisted bio-jetting of hematopoietic stem cells

The research reported in this communication demonstrates the emerging direct cell handling technology now widely referred to as aerodynamically assisted bio-jetting. This is a non-electric field driven approach which directly competes with bio-electrosprays. The technology in these investigations has been explored for the direct handling of live murine primary hematopoietic stem cells. The viability studies demonstrate the complete inertness of this technology for handling such cells for a wide range of applications in both basic biology and clinical medicine. Interestingly these studies pave the way for this technology to undergo development as a flow cell for utility as a sheathless cell most useful in flow cytometry.     

Single molecule study of DNA collision with elliptical nanoposts conveyed by hydrodynamics

Periodic arrays of micro‐ or nanopillars constitute solid‐state matrices with excellent properties for DNA size separation. Nanofabrication technologies offer many solutions to tailor the geometry of obstacle arrays, yet most studies have been conducted with cylinders arranged in hexagonal lattices. In this report, we investigate the dynamics of single DNA collision with elliptical nanoposts using hydrodynamic actuation. Our data show that the asymmetry of the obstacles has minor effect on unhooking dynamics, and thus confirm recent predictions obtained by Brownian dynamics simulations. In addition, we show that the disengagement dynamics are correctly predicted by models of electrophoresis, and propose that this consistency is associated to the confinement in slit‐like channels. We finally conclude that elliptical posts are expected to marginally improve the performances of separation devices.     

Preparation of high specific activity radiotracers for radioanalytical studies

High specific activity radiotracers are very suitable for studies related to the toxicological impact of trace elements onto human health. In order to investigate the metabolic behaviour of Low Level Exposure (LLE) to trace elements, it is necessary to carry out in-vitro and in-vivo experiments with tracers whose concentration is of the same order of magnitude as the present environmental exposure values (ng-g/kg day). Preparations, separations and purifications of some No Carrier Added (NCA) radiotracers (⁴⁸V,^95m,96Tc,^{195m,g,197m,g}Hg,¹⁹⁹Au,²⁰²Tl) produced by either cyclotron or nuclear reactor irradiation, are presented and discussed. This work reviews the more recent advances in this field carried out by our groups.     

In-vivo and In-vitro Metabolism Study of Timosaponin B-II Using HPLC-ESI-MS n

Timosaponin B-II (TB-II), a representative furostanol saponin in Rhizoma anemarrhenae, has been used as an emperor herb in many Chinese herbal formulas to treat diabetes and senile dementia. However, its metabolism and tissue distribution had not been investigated so far. In this work, a sensitive and specific high-performance liquid chromatography-electrospray ionization tandem mass spectrometry method was applied for the identification of TB-II and its major metabolites in in-vivo and in-vitro samples. Rat urine, feces, plasma and tissues were collected after oral administration of TB-II at a single dose of 300 mg kg⁻¹. Furthermore, TB-II was incubated in artificial gastric juice (AGJ) and artificial intestinal juice (AIJ). As a result, 19 metabolites were detected and identified by comparing their HPLC behavior and MSⁿ spectra profile with those of the parent drug. Moreover, the structures of its five metabolites were identified by using the standards prepared by the acid hydrolysis of TB-II. In addition to the parent drug, 14, 12, 6, 1, 1 and 7 metabolites were detected in rat urine, feces, plasma, heart, kidney and liver, respectively, while no metabolites or the parent drug were found in rat brain, spleen and lung. Seven metabolites appeared in AIJ incubation samples, but the parent drug was absent. Nine metabolites along with the parent drug were observed in AGJ incubation samples. The biotransformation pathways of TB-II mainly included dehydration, deglycosylation, hydroxylation, oxidation and E-ring cleavage. This is the first comprehensive investigation of the in-vivo and in-vitro metabolism of TB-II. The result provided important information for further pharmacological research on TB-II.

     

Tetracyclines as a potential antiviral therapy against Crimean Congo hemorrhagic fever virus: Docking and molecular dynamic studies

Crimean-Congo Hemorrhagic Fever Virus (CCHFV) is one of the deadliest human diseases with mortality rate near 50%. Special attention should be paid to this virus since there is no approved treatment for it. On the other hand, the recent outbreak of Ebola virus which is a member of hemorrhagic fever viruses shows this group of viruses can be extremely dangerous. Previous studies have indicated that nucleoprotein of CCHFV, a pivotal protein in virus replication, is an appropriate target for antiviral drug development. The aim of this study is finding inhibitor(s) of this protein. Herein, a virtual screening procedure employing docking followed by molecular dynamic was used to identify small molecule inhibitors of the nucleoprotein from FDA-approved drugs. Regarding CCHFV, using in-silico method is a safe way to achieve its inhibitor(s) since this virus is categorized as a World Health Organization (WHO) biosafety level 4 pathogen and therefore investigation in general laboratories is restricted. In conclusion, considering docking and molecular dynamic results alongside with bioavailability of FDA-approved drugs, doxycycline and minocycline are proposed as potential inhibitors of CCHFV nucleoprotein. There is hope, this study encourage other research groups for in-vitro and in-vivo studies about the efficacy of those two medicines in CCHFV treatment.     

Cell‐Laden Hydrogels for Multikingdom 3D Printing

Living materials are created through the embedding of live, whole cells into a matrix that can house and sustain the viability of the encapsulated cells. Through the immobilization of these cells, their bioactivity can be harnessed for applications such as bioreactors for the production of high‐value chemicals. While the interest in living materials is growing, many existing materials lack robust structure and are difficult to pattern. Furthermore, many living materials employ only one type of microorganism, or microbial consortia with little control over the arrangement of the various cell types. In this work, a Pluronic F127‐based hydrogel system is characterized for the encapsulation of algae, yeast, and bacteria to create living materials. This hydrogel system is also demonstrated to be an excellent material for additive manufacturing in the form of direct write 3D‐printing to spatially arrange the cells within a single printed construct. These living materials allow for the development of incredibly complex, immobilized consortia, and the results detailed herein further enhance the understanding of how cells behave within living material matrices. The utilization of these materials allows for interesting applications of multikingdom microbial cultures in immobilized bioreactor or biosensing technologies.     

Synthesis,biological evaluation and biodistribution of the <Superscript>99m</Superscript>Tc–Garenoxacin complex in artificially infected rats

The labeling of garenoxacin (GXN) with technetium-99m (^99mTc) using different concentrations of GXN, sodium pertechnetate (Na^99mTcO₄), stannous chloride dihydrate (SnCl₂·2H₂O) at different pH was investigated and evaluated in terms of in-vitro stability in saline, serum, binding with multi-resistant Staphylococcus aureus (MDRSA) and penicillin-resistant Streptococci (PRSC) and its biodistribution in artificially MDRSA and PRSC infected rats. ^99mTc–GXN complex with 97.45 ± 0.18% radiochemical stability was prepared by mixing 3 mg of GXN with 3 mCi of Na^99mTcO₄ in the presence of 150 μL of SnCl₂·2H₂O (1 μg/μL in 0.01 N HCl) at a pH 5.6. The radiochemical stability of the complex was evaluated in normal saline up to 240 min of reconstitution. It was observed that the complex showed maximum RCP values after 30 min of the reconstitution and remained more than 90% up to 240 min. The complex showed radiochemical stability in normal saline at 37 °C up to 16 h with a 17.80% de-tagging. The complex showed saturated in-vitro binding with living MDRSA and PRSC as compared to the insignificant binding with heat killed MDRSA and PRSC. Biodistribution behavior of the complex was assessed in artificially infected with living and heat killed MDRSA and PRSC rats. It was observed that the accumulation of the complex in the infected (live MDRSA and PRSC) tissue of the rats was almost five fold than in the inflamed and normal tissue. The high radiochemical stability in normal saline at room temperature, promising in-vitro stability in serum at 37 °C, saturated in-vitro binding with living MDRSA and PRSC, specific biodistribution behavior and high infected (target) to normal (non-target) tissue and low inflamed (non-target) to normal (non-target) tissue ratios we recommend ^99mTc–GXN complex for in-vivo localization of infection caused by MDRSA and PRSC effective stains.     

Application of radiotracers with high specific radioactivity to metallotoxicological studies

The use of radiotracers with high specific activity in research on health impact of trace metals overcomes many of the analytical difficulties associated with experimentation carried out at metal levels which are typical of the polluted environment. It allows one to detect and measure ultratrace amounts of metals in biological samples and to follow them into different biochemical compartments, such as subcellular fractions and molecular components. This work shows typical examples of metallotoxicological studies carried out at the JRC-Ispra using radioisotopes with high specific radiactivity produced in the nuclear reactor and in the cyclotron. Applications refer to the use of⁴⁵Ti,⁴⁸V,⁶⁴Cu,⁹⁵Nb and^106mAg in in-vivo and in-vitro studies related to environmental and occupational toxicology research on trace metals.