Gene electrotransfer: from biophysical mechanisms to in vivo applications: Part 1- Biophysical mechanisms

Electropulsation is one of the nonviral methods successfully used to deliver genes into living cells in vitro and in vivo. This approach shows promise in the field of gene and cellular therapies. The present review focuses on the processes supporting gene electrotransfer in vitro. In the first part, we will report the events occurring before, during, and after pulse application in the specific field of plasmid DNA electrotransfer at the cell level. A critical discussion of the present theoretical considerations about membrane electropermeabilization and the transient structures involved in the plasmid uptake follows in a second part.     

In vivo biosensors: mechanisms,development, and applications

In vivo biosensors can recognize and respond to specific cellular stimuli. In recent years, biosensors have been increasingly used in metabolic engineering and synthetic biology, because they can be implemented in synthetic circuits to control the expression of reporter genes in response to specific cellular stimuli, such as a certain metabolite or a change in pH. There are many types of natural sensing devices, which can be generally divided into two main categories: protein-based and nucleic acid-based. Both can be obtained either by directly mining from natural genetic components or by engineering the existing genetic components for novel specificity or improved characteristics. A wide range of new technologies have enabled rapid engineering and discovery of new biosensors, which are paving the way for a new era of biotechnological progress. Here, we review recent advances in the design, optimization, and applications of in vivo biosensors in the field of metabolic engineering and synthetic biology.     

In vivo gene expression: combining hydrodynamics-based transfection and electrotransfer

Clinical development of gene therapy is hampered by the lack of an efficient and safe method for in vivo gene transfer. New methodologies for plasmid transfer are being developed. Recently, significant expression of a reporter gene was achieved in liver cells by coupling intravenous injection and stimulation of the tissue with electric pulses. This combination of hydrodynamics-based transfection and electrotransfer could provide the basis for many promising clinical applications.     

Gene delivery mediated by cationic liposomes: from biophysical aspects to enhancement of transfection

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. However, the relationship between the features of the lipid-DNA complexes (`lipoplexes') and their mode of interaction with cells, the efficiency of gene transfer and gene expression remain to be clarified. To gain insights into these aspects, the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3- (trimethylammonium) propane (DOTAP) and its mixture with phosphatidylethanolamine (PE)), and their complexes with DNA at different (+/-) charge ratios were determined. A lipid mixing assay was used to assess the interaction of liposomes and lipoplexes with monocytic leukaemia cells. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. However, very limited transfection of these cells was achieved using the above complexes. It is possible that the topology of the cationic liposome-DNA complexes does not allow the entry of DNA into cells through a fusion process at the plasma membrane. In an attempt to enhance transfection mediated by lipoplexes composed of DOTAP and its equimolar mixture with dioleoylphosphatidylethanolamine (DOPE) two different strategies were explored: (i) association of a targeting ligand (transferrin) to the complexes to promote their internalization, presumably by receptor-mediated endocytosis; and (ii) association of synthetic fusogenic peptides (GALA or the influenza haemagglutinin Nterminal peptide HA-2) to the complexes to promote endosomal destabilization and release of the genetic material into the cytoplasm. These strategies were effective in enhancing transfection in a large variety of cells, including epithelial and lymphoid cell lines, as well as human macrophages, especially with the use of optimized lipid/ DNA (+/-) charge ratios. Besides leading to high levels of transfection, the ternary complexes of cationic liposomes, DNA, and protein or peptide, have the advantages of being active in the presence of serum and being non-toxic. Moreover, such ternary complexes present a net negative charge and, thus, are likely to alleviate the problems associated with the use of highly positively charged complexes in vivo, such as avid complexation with serum proteins. Overall, the results indicate that these complexes, and their future derivatives, may constitute viable alternatives to viral vectors for gene delivery in vivo.     

Gene editing: from technologies to applications in research and beyond

Science China Life Sciences -     

Gene delivery mediated by cationic liposomes: from biophysical aspects to enhancement of transfection.

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. However, the relationship between the features of the lipid-DNA complexes ('lipoplexes') and their mode of interaction with cells, the efficiency of gene transfer and gene expression remain to be clarified. To gain insights into these aspects, the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3- (trimethylammonium) propane (DOTAP) and its mixture with phosphatidylethanolamine (PE)), and their complexes with DNA at different (+/-) charge ratios were determined. A lipid mixing assay was used to assess the interaction of liposomes and lipoplexes with monocytic leukaemia cells. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. However, very limited transfection of these cells was achieved using the above complexes. It is possible that the topology of the cationic liposome-DNA complexes does not allow the entry of DNA into cells through a fusion process at the plasma membrane. In an attempt to enhance transfection mediated by lipoplexes composed of DOTAP and its equimolar mixture with dioleoylphosphatidylethanolamine (DOPE) two different strategies were explored: (i) association of a targeting ligand (transferrin) to the complexes to promote their internalization, presumably by receptor-mediated endocytosis; and (ii) association of synthetic fusogenic peptides (GALA or the influenza haemagglutinin N-terminal peptide HA-2) to the complexes to promote endosomal destabilization and release of the genetic material into the cytoplasm. These strategies were effective in enhancing transfection in a large variety of cells, including epithelial and lymphoid cell lines, as well as human macrophages, especially with the use of optimized lipid/DNA (+/-) charge ratios. Besides leading to high levels of transfection, the ternary complexes of cationic liposomes, DNA, and protein or peptide, have the advantages of being active in the presence of serum and being non-toxic. Moreover, such ternary complexes present a net negative charge and, thus, are likely to alleviate the problems associated with the use of highly positively charged complexes in vivo, such as avid complexation with serum proteins. Overall, the results indicate that these complexes, and their future derivatives, may constitute viable alternatives to viral vectors for gene delivery in vivo.     

Polysaccharide-coated thermosets for orthopedic applications: from material characterization to in vivo tests

The long-term stability and success of orthopedic implants depend on the osseointegration process, which is strongly influenced by the biomaterial surface. A promising approach to enhance implant integration involves the modification of the surface of the implant by means of polymers that mimic the natural components of the extracellular matrix, for example, polysaccharides. In this study, methacrylate thermosets (bisphenol A glycidylmethacrylate/triethyleneglycol dimethacrylate), a widely used composition for orthopedic and dental applications, have been coated by electrostatic deposition of a bioactive chitosan-derivative. This polysaccharide was shown to induce osteoblasts aggregation in vitro, to stimulate cell proliferation and to enhance alkaline phosphatase activity. The coating deposition was studied by analyzing the effect of pH and ionic strength on the grafting of the polysaccharide. Contact angle studies show that the functionalized material displays a higher hydrophilic character owing to the increase of surface polar groups. The mechanical properties of the coating were evaluated by nanoindentation studies which point to higher values of indentation hardness and modulus (E) of the polysaccharide surface layer, while the influence of cyclic stress on the construct was assessed by fatigue tests. Finally, in vivo tests in minipigs showed that the polysaccharide-based implant showed a good biocompatibility and an ability for osseointegration at least similar to that of the titanium Ti6Al4V alloy with roughened surface.     

Radiative decay engineering: biophysical and biomedical applications.

Fluorescence spectroscopy is a widely used research tool in biochemistry and molecular biology. Fluorescence has also become the dominant method enabling the revolution in medical diagnostics, DNA sequencing, and genomics. To date all the fluorescence observables, including spectral shifts, anisotropies, quantum yields, and lifetimes, have all been utilized in basic and applied uses of fluorescence. In this forward-looking article we describe a new opportunity in fluorescence, radiative decay engineering (RDE). By RDE we mean modifying the emission of fluorophores or chromophores by increasing or decreasing their radiative decay rates. In most fluorescence experiments the radiative rates are not changed because these rates depend on the extinction coefficient of the fluorophore. This intrinsic rate is not changed by quenching and is only weakly dependent on environmental effects. Spectral changes are usually caused by changes in the nonradiative rates resulting from quenching or resonance energy transfer. These processes affect the emission by providing additional routes for decay of the excited states without emission. In contrast to the relatively constant radiative rates in free solution, it is known that the radiative rates can be modified by placing the fluorophores at suitable distances from metallic surfaces and particles. This Review summarizes results from the physics literature which demonstrate the effects of metallic surfaces, colloids, or islands on increasing or decreasing emissive rates, increasing the quantum yields of low quantum yield chromophores, decreasing the lifetimes, and directing the typically isotropic emission in specific directions. These effects are not due to reflection of the emitted photons, but rather as the result of the fluorophore dipole interacting with free electrons in the metal. These interactions change the intensity and temporal and spatial distribution of the radiation. We describe the unusual effects expected from increases in the radiative rates with reference to intrinsic and extrinsic biochemical fluorophores. For instance, the decreased lifetime can result in an effective increase in photostability. Proximity to nearby metallic surfaces can also increase the local field and modify the rate of excitation. We predict that the appropriate localization of fluorophores near particles can result in usefully high emission from "nonfluorescent" molecules and million-fold increases in the number of photons observable from each fluorophore. We also describe how RDE can be applied to medical testing and biotechnology. As one example we predict that nearby metal surfaces can be used to increase the low intrinsic quantum yields of nucleic acids and make unlabeled DNA detectable using its intrinsic metal-enhanced fluorescence.     

In vivo imaging of free radicals: applications from mouse to man

Free radicals and other paramagnetic species, play an important role in cellular injury and pathophysiology. EPR spectroscopy and imaging has emerged as an important tool for non-invasive in vivo measurement and spatial mapping of free radicals in biological tissues. Extensive applications have been performed in small animals such as mice and recently applications in humans have been performed. Spatial EPR imaging enables 3D mapping of the distribution of a given free radical while spectral-spa-tial EPR imaging enables mapping of the spectral information at each spatial position, and, from the observed line width, the localized tissue oxygenation can be determined. A variety of spatial, and spectral-spatial EPR imaging applications have been performed. These techniques, along with the use of biocompatible paramagnetic probes including particulate suspensions and soluble nitroxide radicals, enable spatial imaging of the redox state and oxygenation in a variety of biomedical applications. With spectral-spatial EPR imaging, oxygenation was mapped within the gastrointestinal (GI) tract of living mice, enabling measurement of the oxygen gradient from the proximal to the distal GI tract. Using spatial EPR imaging, the distribution and metabolism of nitroxide radicals within the major organs of the body of living mice was visualized and anatomically co-registered by proton MRI enabling in vivo mapping of the redox state and radical clearance. EPR imaging techniques have also been applied to non-invasively measure the distribution and metabolism of topically applied nitroxide redox probes in humans, providing information regarding the penetration of the label through the skin and measurement of its redox clearance. Thus, EPR spectroscopy and imaging has provided important information in a variety of applications ranging from small animal models of disease to topical measurement of redox state in humans.     

Linking the computational structure of variance adaptation to biophysical mechanisms

In multiple sensory systems, adaptation to the variance of a sensory input changes the sensitivity, kinetics, and average response over timescales ranging from < 100 ms to tens of seconds. Here, we present a simple, biophysically relevant model of retinal contrast adaptation that accurately captures both the membrane potential response and all adaptive properties. The adaptive component of this model is a first-order kinetic process of the type used to describe ion channel gating and synaptic transmission. From the model, we conclude that all adaptive dynamics can be accounted for by depletion of a signaling mechanism, and that variance adaptation can be explained as adaptation to the mean of a rectified signal. The model parameters show strong similarity to known properties of bipolar cell synaptic vesicle pools. Diverse types of adaptive properties that implement theoretical principles of efficient coding can be generated by a single type of molecule or synapse with just a few microscopic states.     

On the electrotransfer of polypeptides from gels to nitrocellulose membranes

The conditions which affect the elution of polypeptides from polyacrylamide gels by electrophoresis and polypeptide-nitrocellulose interactions have been studied. The rate of elution of polypeptides from a 15% sodium dodecyl sulfate-polyacrylamide gel is dependent on the molecular weight of the individual polypeptides, which is in agreement with the results of W. N. Burnette (Anal. Biochem. 112, 195 (1981)). We also observed that current density affects the rate of elution. Polypeptides smaller than 20,000 daltons pass through pores of 0.45 microns, but not through the pores of 0.1-microns nitrocellulose membranes during electrophoresis. The nonionic detergent NP-40 inhibits the binding of polypeptides to nitrocellulose and removes prebound polypeptides from the membranes. Amido black and Coomassie blue staining and destaining processes do not remove the bound polypeptides from the membranes, but may affect the antigenicity of polypeptides. Polypeptides immobilized on nitrocellulose can be stored at -70 degrees C for future use.     

IRIV-adjuvanted hepatitis A vaccine: in vivo absorption and biophysical characterization

Immunopotentiating reconstituted influenza virosomes (IRIV) are 150-nm proteoliposomes composed of influenza surface glycoproteins and a mixture of natural and synthetic phospholipids. Due to size, structure and composition of the IRIVs, they serve as an antigen carrier system for efficacious vaccination, as was demonstrated for hepatitis A and influenza. This paper reviews the unique properties of IRIVs and describes the in vivo biodistribution of model antigens using 14C-labeled IRIVs and 125I-labeled streptavidin. IRIV formulated streptavidin induced a strong depot effect after intra muscular (i.m.) vaccination of mice, whereas soluble streptavidin was soon eliminated via the kidney of the animals. A mixture of antigen and IRIVs yielded higher antibody titers after i.m. inoculation than streptavidin alone. The highest immunostimulation was achieved by the binding of the antigen to the investigated adjuvant. The potential penetration of inactivated hepatitis A virions into lipid membranes was assessed by measuring the area increase of a lipid monolayer kept at a constant surface pressure corresponding to that of lipid bilayer vesicles. The monolayers were composed of phosphatidylcholine (POPC) and phosphatidylethanolamine (POPE) (75/25 mol/mol), thus resembling the lipid composition of the IRIV. The results suggested that the hepatitis A antigen may spontaneously bind to the reconstituted IRIV membranes.