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Electroporation has been a widely established method for delivering DNA and other material into cells in vitro. Conventional electroporation infrastructure is typically immobile, non-customizable, non-transparent regarding the characteristics of output pulses, and expensive. Here, we describe a portable electroporator for DNA delivery into bacterial cells that can quickly be reconstructed using 3D desktop printing and off-the-shelf components. The device is light weight (700 g), small (70 × 180 × 210 mm) and extremely low-cost (

Fluorescence Correlation Spectroscopy (FCS) is a promising tool to study interactions on a single molecule level. The diffusion of fluorescent molecules in and out of the excitation volume of a confocal microscope leads to the fluorescence fluctuations that give information on the average number of fluorescent molecules present in the excitation volume and their diffusion coefficients. In this context, we complexed mRNA into lipoplexes and polyplexes and explored the association/dissociation degree of complexes by using gel electrophoresis and FCS. FCS enabled us to measure the association and dissociation degree of mRNA-based complexes both in buffer and protein-rich biological fluids such as human serum and ascitic fluid, which is a clear advantage over gel electrophoresis that was only applicable in protein-free buffer solutions. Furthermore, following the complex stability in buffer and biological fluids by FCS assisted to understand how complex characteristics, such as charge ratio and strength of mRNA binding, correlated to the transfection efficiency. We found that linear polyethyleneimine prevented efficient translation of mRNA, most likely due to a too strong mRNA binding, whereas the lipid based carrier Lipofectamine® messengerMAX did succeed in efficient release and subsequent translation of mRNA in the cytoplasm of the cells. Overall, FCS is a reliable tool for the in depth characterization of mRNA complexes and can help us to find the critical balance keeping mRNA bound in complexes in the extracellular environment and efficient intracellular mRNA release leading to protein production. Statement of Significance The delivery of messenger RNA (mRNA) to cells is promising to treat a variety of diseases. Therefore, the mRNA is typically packed in small lipid particles or polymer particles that help the mRNA to reach the cytoplasm of the cells. These particles should bind and carry the mRNA in the extracellular environment (e.g. blood, peritoneal fluid, …), but should release the mRNA again in the intracellular environment. In this paper, we evaluated a method (Fluorescence Correlation Spectroscopy) that allows for the in depth characterization of mRNA complexes and can help us to find the critical balance keeping mRNA bound in complexes in the extracellular environment and efficient intracellular mRNA release leading to protein production.

Mixtures of surfactants can result in formation of various structures like micelles, vesicles and inverted micelles. Catanionic vesicular systems are preferred on account of their ease of formation and thermodynamic stability. Furthermore, their charge and surfactant properties render them as useful vehicles for DNA delivery and cytotoxic compounds. They suffer from disadvantages of being leaky and yielding low encapsulation efficiencies which are averse to drug delivery purposes. Extensive efforts are being undertaken to overcome these barriers and render these vesicles amenable to spatial placement and temporal delivery of drugs. This manuscript addresses diverse aspects of catanionic vesicles including their formation, fabrication and stability. The manuscript focuses further on applications of catanionic vesicles in nanodrug delivery. Novel trends in the field of catanionics with respect to bio-compatibility and novel technologies developed using these systems have also been reviewed. An attempt has been made to compile catanionic systems reported in literature detailing surfactants and therapeutic agents employed to aid understanding and yield information of various facets that drive fabrication and potential utility of these systems in therapeutics.

DNA vaccines are ideal candidates for global vaccination purposes because they are inexpensive and easy to manufacture on a large scale such that even people living in low-income countries can benefit from vaccination. However, the potential of DNA vaccines has not been realized owing mainly to the poor cellular uptake of DNA in vivo resulting in the poor immunogenicity of DNA vaccines. In this review, we discuss the benefits and shortcomings of several promising and innovative non-biological methods of DNA delivery that can be used to increase cellular delivery and efficacy of DNA vaccines.

In the last years, vaginal gene therapy has gained increasing attention mainly for the treatment and control of sexually transmitted infections. DNA delivery has been also suggested to improve reproductive outcomes for women with deficiencies in the female reproductive tract. Although no product has reached clinical phase, preclinical investigations reveal the potential of the vaginal tract as an effective administration route for gene delivery. This review focuses on the main advantages and challenges of vaginal gene therapy, and on the most used nucleic acid delivery systems, including viral and non-viral vectors. Additionally, the advances in the application of vaginal gene therapy for the treatment and/or prevention of infectious diseases such as the human immunodeficiency virus (HIV), the human papillomavirus (HPV) or the herpes simplex virus (HSV) are presented.

Microfluidics has recently emerged as a new method of manufacturing liposomes, which allows for reproducible mixing in miliseconds on the nanoliter scale. Here we investigate microfluidics-based manufacturing of liposomes. The aim of these studies was to assess the parameters in a microfluidic process by varying the total flow rate (TFR) and the flow rate ratio (FRR) of the solvent and aqueous phases. Design of experiment and multivariate data analysis were used for increased process understanding and development of predictive and correlative models. High FRR lead to the bottom-up synthesis of liposomes, with a strong correlation with vesicle size, demonstrating the ability to in-process control liposomes size; the resulting liposome size correlated with the FRR in the microfluidics process, with liposomes of 50nm being reproducibly manufactured. Furthermore, we demonstrate the potential of a high throughput manufacturing of liposomes using microfluidics with a four-fold increase in the volumetric flow rate, maintaining liposome characteristics. The efficacy of these liposomes was demonstrated in transfection studies and was modelled using predictive modeling. Mathematical modelling identified FRR as the key variable in the microfluidic process, with the highest impact on liposome size, polydispersity and transfection efficiency. This study demonstrates microfluidics as a robust and high-throughput method for the scalable and highly reproducible manufacture of size-controlled liposomes. Furthermore, the application of statistically based process control increases understanding and allows for the generation of a design-space for controlled particle characteristics.

The purpose of this study was to evaluate a motorized microneedle device in delivery of DNA into skin for gene expression. A plasmid DNA encoding both luciferase (Luc) and enhanced green fluorescent protein (EGFP) was delivered into rat skin by puncturing the skin with the microneedle device. Puncturing rat skin with a pre-applied DNA solution on the skin showed much higher luciferase gene expression than that with the procedure of puncturing the skin first then applied the DNA solution. The microneedle puncturing method was more efficient than intradermal injection method in generating high gene expression in the skin. There was no significant difference in the skin gene expression when rat skin was punctured with the microneedle device of different microneedle lengths (0.25 mm, 0.5mm or 0.75 mm). On the other hand, there was a significant difference in the skin gene expression between the short (10s) and the long puncturing durations (30 or 60s), with longer puncturing duration showed higher gene expression. Puncturing the skin with longer needles (0.75 mm) caused some skin damage, while puncturing the skin with shorter microneedle length (0.25 mm) caused only minimal skin damage. The EGFP gene expression was observed predominately in the epidermis layer of the skin from the puncturing method in delivery of DNA into the skin. In summary, the motorized microneedle device could have great potential in skin gene delivery.

This paper describes the synthesis of a combinatorial library of quaternized lipidoids (QLDs) and an evaluation of their abilities to facilitate in vitro DNA delivery. The QLDs alone showed low efficiency for DNA delivery. By formulating liposomes with a neutral helper lipid, such as 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine (DOPE), the capability of QLDs for gene transfection is significantly enhanced due to the fusogenic properties of DOPE which facilitate endosomal escape and cargo delivery. We further optimized the liposome composition and DNA dose for gene transfection and investigated the structure–activity relationships of the lipidoid library in DNA delivery. From the Clinical Editor This paper describes the synthesis and evaluation of a combinatorial library of quaternized lipidoids to facilitate in vitro DNA delivery, which occurs at a low level but can be enhanced with DOPE. The authors also further optimized the liposome composition and DNA dose for delivery and investigated the structure-activity relationships of the lipidoid library.

Gene therapy offers great opportunities for the treatment of severe diseases including cancer. In recent years the design of synthetic carriers for nucleic acid delivery has become a research field of increasing interest. Studies on the delivery of plasmid DNA (pDNA) have brought up a variety of gene delivery vehicles. The more recently emerged gene silencing strategy by the intracellular delivery of small interfering RNA (siRNA) takes benefit from existing expertise in pDNA transfer. Despite common properties however, delivery of siRNA also faces distinct challenges due to apparent differences in size, stability of the formed nucleic acid complexes, the location and mechanism of action. This review emphasizes the common aspects and main differences between pDNA and siRNA delivery, taking into consideration a wide spectrum of polymer-based, lipidic and peptide carriers. Challenges and opportunities which result from these differences as well as the recent progress made in the optimization of carrier design are presented.

Development of materials with stimuli-responsive properties is of interest for biotechnical applications including gene delivery and regenerative medicine. Here, we report a multilayer film through layer-by-layer self-assembly of DNA polyplexes and charge-reversible poly(ethylenimine) (cPEI). Through functionalizing PEI with cyclohexanedicarboxylic acid, cPEI showed negatively charged and therefore was used for electrostatic self-assembly with positively charged DNA polyplexes. Side chains of cPEI can be hydrolyzed in acidic environment while it is stable in neutral condition. Such pH-triggered hydrolysis led to charge reverse of cPEI from negative to positive, which consequently led to a disassembly of multilayer film. Both UV–vis and ellipsometry spectrum measurements suggested that the multilayer film grew with a thickness of 150 nm for twelve bilayers. Under low pH condition, the multilayer film collapsed and DNA polyplexes were released. The multilayer film containing cPEI could be served as a local gene delivery system in specific low pH conditions such as extracellular acidity of solid tumor and lysosomal.

We report the effects of a synthetic peptide designed to act as a nuclear localization signal on the treatment of tuberculosis. The peptide contains 21 amino acid residues with the following specific domains: nuclear localization signal from SV 40T, cationic shuttle sequence, and cysteamide group at the C-terminus. The peptide was complexed with the plasmid DNAhsp65 and incorporated into cationic liposomes, forming a pseudo-ternary complex. The same cationic liposomes, composed of egg chicken l-α-phosphatidylcholine, 1,2-dioleoyl-3-trimethylammonium-propane, and 1,2-dioleoyl-3-trimethylammonium-propane (2:1:1M), were previously evaluated as a gene carrier for tuberculosis immunization protocols with DNAhsp65. The pseudo-ternary complex presented a controlled size (250nm), spherical-like shape, and various lamellae in liposomes as evaluated by transmission electron microscopy. An assay of fluorescence probe accessibility confirmed insertion of the peptide/DNA into the liposome structure. Peptide addition conferred no cytotoxicity in vitro, and similar therapeutic effects against tuberculosis were seen with four times less DNA compared with naked DNA treatment. Taken together, the results indicate that the pseudo-ternary complex is a promising gene vaccine for tuberculosis treatment. This work contributes to the development of multifunctional nanostructures in the search for strategies for in vivo DNA delivery.

Hollow deoxyribonucleic acid (DNA)/poly-L-lysine (PLL) capsules were successfully fabricated through a layer-by-layer (LbL) self-assembly of DNA and PLL on porous CaCO3 microparticles, followed by removal of templates with ethylenediamine tetraacetic acid disodium salt (EDTA). The enzymatic degradation of the capsules in the presence of alpha-chymotrypsin was explored. The higher the enzyme concentration, the higher is the degradation rate of hollow capsules. in addition, glutaric dialdehyde (GA) cross-linking was found to be another way to manipulate degradation rate of hollow capsules.

In many bacteria, gene replacement can only be achieved using RP4-based conjugative systems where exogenous DNA is delivered to the recipient on conditionally replicating plasmids carrying an oriT RP4 . In the commonly used strains SM10 and S17-1 conjugative functions are provided by an RP4 plasmid inserted in the chromosome. Those strains, besides mobilizing oriT RP4 -carrying plasmids, also transfer their own chromosomal genes, which can be inconvenient for several reasons. We describe here a new version of an RP4-based conjugative system that allows for efficient transfer of mobilizable plasmids without the transfer of chromosomal genes, providing fully controlled and efficient exogeneous DNA delivery to non-transformable bacteria.

Gene therapy has long sought to treat disease by delivering therapeutic genes to diseased cells. However, its widespread clinical use has faltered in the face of several challenges, most notably in the area of delivery. Efforts to understand the biological barriers that prevent the effective delivery of therapeutic genes to targeted tissues, and developing safe and efficient vectors that can overcome the barriers, stand at the heart of current gene delivery research. The first part of this review provides a comprehensive discussion of the current understanding of these biological barriers and the rich collection of polymers and design strategies that have accumulated from nearly two decades of research aimed at overcoming these hurdles. The second part highlights the challenges that remain and several innovative approaches and methods that have recently been developed representing, in part, the future directions of gene delivery research.

Since compounds targeting mitochondria exhibit diverse accumulation mechanisms and chemical features, various questions arise. Do such “mitochondriotropics” have a characteristic chemistry? What are mitochondrial uptake mechanisms? Do mitochondriotropics necessarily accumulate in mitochondria or merely have access? Is the concept “mitochondriotropic” of any practical value? To seek answers, a non-biased sample of > 100 mitochondriotropics was generated from the review literature. This dataset was examined using: physicochemical classification; quantitative structure-activity relations (QSAR) models; and a Fick–Nernst–Planck physicochemical model. The ability of the latter two approaches to predict mitochondriotropic behaviour was assessed, and comparisons made between methods, and with current assumptions. All approaches provided instructive pictures of the nature of mitochondriotropics. Thus although lipophilic cations are regarded as the commonest structural type, only a third were such. Much the same proportion were acids, potentially or actually anions. Many mitochondriotropics were electrically neutral compounds. All categorizations involved overall molecular properties, not the presence of “mitochondriotropic tags” — again contrary to literature concepts. Selective mitochondrial accumulation involved electric potential, ion-trapping, and complex formation with cardiolipin; non-specific accumulation involved membrane partitioning. Non-specific access required only low lipophilicity. Mitochondrial targeting did not preclude additional accumulation sites, e.g. lysosomes. The concept “mitochondriotropic” remains useful, although may imply access, not accumulation. QSAR and Fick–Nernst–Planck approaches are complementary — neither is universally applicable. Using both approaches enabled the mitochondriotropic behavior of > 80% of the dataset to be predicted, and the physicochemistry of mitochondriotropics to be specified in some detail. This can facilitate guided syntheses and selection of optimal mitochondriotropic structures.

Through the layer-by-layer (LbL) self-assembly technique, DNA was incorporated into the multilayered films with poly-l-lysine (PLL). The effect of ionic strength on the construction and deconstruction of the PLL/DNA films was investigated. It was found that the salt concentration of the deposition solution had a significant effect on the construction of the films, which might attribute to the effect of salt ions on the conformation of polyelectrolytes and interaction between PLL and DNA molecules. A salt-induced deconstruction of the PLL/DNA films was observed. The extent of the deconstruction increased with the salt concentration in the incubation solution. The mechanism of the deconstruction was discussed. Taking the advantages of the LbL technique, the erasable PLL/DNA films could deposit onto a variety of surfaces, such as vascular stent, intervention catheter and tissue engineering scaffold, to serve as a novel DNA delivery system.

We have characterized the impact of strain-based genetic differences on the efficiency of the intravenous cationic liposome-DNA complex (CLDC)-based gene transfer and expression in mice. We also investigated what steps in the gene delivery and expression pathway appeared responsible for these strain-related differences and whether such differences could be compensated for either by agents that alter host pathways important in CLDC-mediated gene transfer and expression, or by changes in CLDC formulation. We found that different mouse strains can exhibit different expression levels and/or differences in the amount of plasmid DNA delivered to the organs where the DNA is expressed. Furthermore, drug pretreatment or reformulation of the CLDC could improve DNA delivery and/or gene expression in a strain-specific fashion. We conclude that genetic factors critically modify both the tissue deposition and the expression of genetic materials delivered by CLDC. Because manipulation of either the host or the CLDC could at least partially compensate for these strain-related differences, such strategies may be required to effectively use non-viral gene transfer approaches in genetically diverse populations.

Chinese Acad Sci, Inst Proc Engn, State Key Lab Biochem Engn, Beijing 100190, Peoples R China

Purpose: To evaluate a single institutional experience with minimally invasive limited ligation endoluminal-assisted revision (MILLER) for treatment of dialysis access steal syndrome (DASS). Materials and Methods: Thirty-three patients (20 men and 13 women; average age 64.4 14.8 years) were retrospectively identified that underwent 46 MILLER procedures for dialysis access steal syndrome and high-output heart failure at our institution from March 2010 to September 2013. Technical success was defined by successful creation of MILLER band without complications. Clinical success was defined as no repeat interventions for 1 month after the initial banding procedure. Results: Technical success was achieved in all patients. Clinical success was reached in 91%. Primary patency rates of MILLER bands created in this study were 91% at 1 month and 76% at 6 months. Conclusion: MILLER banding is a suitable alternative for treatment of DASS.

Patients with progressive kidney disease develop anemia due to declining erythropoietin (EPO) production in the kidneys. The condition is currently treated by the frequent injection of recombinant human EPO. The expression of EPO following gene delivery to an ectopic site could provide a more convenient and more economical long-term solution. The major obstacles for the gene therapy of anemia have been: (1) finding a safe and efficient DNA delivery method to a tissue that can secrete EPO into the blood and (2) controlling the level of EPO expression by some means in order to prevent polycythemia.

Several studies have shown that polycations bind and compact plasmid DNA (pDNA) into colloidal nanoparticles that are readily endocytosed by many cell types with varying degrees of delivery efficiency and toxicity. However, three important issues have arisen with polymer-mediated gene delivery: i) some of the most efficient nonviral agents exhibit cytotoxicity, ii) polymeric vectors have lower DNA delivery efficiency than viral vectors, and iii) the chemical structure of polymeric delivery agents affects the toxicity and delivery efficiency to a large degree.

We, and other have previously shown that using optimised formulations adenovirus (Ad) is several logs more efficient in myocardial gene transfer than “naked” plasmid DNA (pDNA). Here, we evaluated “magnetofection”, the use of magnetic force acting on vectors complexed to magnetic particles and ultrasound to increase non-viral gene transfer to the murine myocardium. C57BL6/J mice were intubated and a thoracotomy performed to expose the heart. The ability of the magnetic force to pass through tissue was tested ex vivo on a dissected lamb's heart. Iron filings sprinkled on the endocardial site moved and clustered together when the magnet was placed on the epicardial side of the left myocardial wall (n=3). A variety of magnetic particles have been described, here we used TransMAGPEI (Chemicell, Germany), a polyethyleneimine (PEI)–coated iron oxide particle. For the myocardial injection the total volume could not exceed 10 μl. The magnetic particle:DNA complexes were carefully optimised by altering the TransMAGPEI to pDNA w/w and v/v ratios to avoid precipitation. The complexes used for in vivo injections were made in the following way: 0.8 μl TransMAGPEI (60 μg/μl) was diluted with 41.7 μl water and mixed with 5 μl pDNA (5 μg/μl) carrying a luciferase reporter gene (pLux). 2.5 μl 0.9% NaCl (18%) was added after 15 minutes. 10 μl of this formulation containing 5 μg pLux were injected into the left ventricular wall. The hearts were exposed to the magnet (AlNiCo Rod magnet plus NeoDelta magnet, IBS) for 5 min straight after injection and for a further 10 min after re-sutering of the chest. The left ventricular wall was harvested 48 hrs after gene transfer and lux expression was measured. Although magnet exposed hearts expressed more lux than the non-magnet exposed heart, this difference was not significant (magnet: 268±124 RLU/mg protein, no magnet: 94.0±41.8 RLU/mg protein, n=8-10/group). It has previously been shown that ultrasound (US) enhances non-viral gene transfer in skeletal muscle probably through acoustic cavitation of the cell membrane. Here, US was applied for 5 minutes before and after myocardial injection of 10 μg pLux in 10 μl total volume containg 1:1 v/v Opitson microbubbles. For this purpose the chest cavity was filled with gel (Henleys Medical, Welwyn Garden City, UK) and a 0.5 cm2 probe massaged over the heart area. The Medi-Link system machine (Electro-Medical Supplies Ltd, Wantage, UK) was set to deliver 1MHz, 2 W/cm2 at 100% duty cycle continous wave. 48 hrs after gene transfer the left vetricular wall was harvested and lux expression measured. In a parallel experiment we demonstrated that ultrasound imaging (14 MHz) using a gel filled chest cavity is feasible and therefore concluded that ultrasound waves were reaching the heart. However, the US conditions described above did not increase lux expression in the mouse heart (US: 71.8±38.4 RLU/mg protein, no US:138±69.2 RLU/mg protein, n=10/group). In conclusion, although physical energy may be useful in improving non-viral gene transfer to the myocardium further optimisation of these techniques will be required.