Search

The interplay of condensation and decondensation of DNA plays a crucial role in chromosome maintenance and gene expression. The molecular architectonics governing the chromatin condensation-decondensation cycle are worth studying, as DNA performs unique and distinct roles in each state and switches between two states without the loss of structural and functional integrity. This phenomenon has been adapted and implemented in transfection studies. Effective gene delivery into the cells to achieve respectable transfection efficiency has remained a challenge and emphasizes the need for understanding the steps involved in DNA delivery and transfection. Especially, recognizing the factors that effectively regulate DNA decondensation can provide logical solutions to the hurdles affecting the transfection efficiency. We designed a set of small molecule-based threading intercalation ligands as model condensing agents to study various factors influencing the DNA condensation and decondensation process. This study revealed condensation of DNA into nanocondensate by the threading intercalator and endogenous stimuli induced effective decondensation. Further, DNA nanocondensates are tracked using the intrinsic fluorescence in the lower pH of endocytic pathway and were evaluated as nonviral vectors for in cellulo delivery of plasmids. The correlation of decondensation of DNA nanocondensate with endogenous metal ions at their physiological concentrations provided valuable insights and implications for intracellular DNA delivery.

Synthetic polycations are studied extensively as DNA delivery agents because of their ease of production, good chemical stability, and low cost relative to viral vectors. This report describes the synthesis of charge-shifting polycations based on N,N-(dimethylamino)ethyl acrylate (DMAEA) and 3-aminopropylmethacryamide (APM), called PAD copolymers, and their use for in vitro DNA delivery into HeLa cells. PAD copolymers of varying compositions were prepared by RAFT polymerization to yield polymers of controlled molecular weights with low dispersities. Model hydrolysis studies were carried out to assess the rate of charge-shifting of the polycations by loss of the cationic dimethylaminoethanol side chains. They showed reduction in the net cationic charge by about 10-50% depending on composition after 2 days at pH 7, forming polyampholytes comprising permanent cationic groups, residual DMAEA, as well as anionic acrylic acid groups. HeLa cells exposed for 4 h to PAD copolymers with the greatest charge-shifting ability showed comparable or higher viability at high concentrations, relative to the noncharge shifting polycations PAPM and polyethyleneimine (PEI) 2 days post-exposure. Cell uptake efficiency of PAD/60bp-Cy3 DNA polyplexes at 2.5:1 N/P ratio was very high (>95%) for all compositions, exceeding the uptake efficiency of PEI polyplexes of equivalent composition. These results suggest that these PAD copolymers, and in particular PAD80 containing 80 mol % DMAEA, have suitable rates of charge-shifting hydrolysis for DNA delivery, as PAD80 showed reduced cytotoxicity at high concentrations, while still retaining high uptake efficiencies. In addition, the polyampholytes formed during DMAEA hydrolysis in PAD copolymers can offer enhanced long-term cytocompatibility.

Background The choice of dialysate sodium (DNa) for haemodialysis (HD) patients remains controversial, with some studies reporting that a lower DNa improves blood pressure control and reduces intradialytic weight gain. Studies on DNa depend on the alignment of programmed to delivered DNa. We wished to determine whether there were differences between programmed and delivered DNa. Methods Dialysate samples were obtained from three dialysis machines: Fresenius 4008H (F4008H) and 5008S (F5008S) and B-Braun hemodiafiltration (HDF) Dialog+(BB). DNa was measured by indirect ion-selective electrode (ISE), flame photometry (FP) and ion chromatography (IC) at different DNa concentrations. Results We tested 18 F5008S, 18 F4008H and 31 BB machines over 153 HD treatments. The median measured minus programmed DNa was significantly greater with the BB machine [ISE, 7 (6–8); FP, 7 (6–8); IC, 6 (5–7)], followed by the F4008H [ISE, 5.5 (5–7); FP, 4 (2.25–5.75); IC, 4 (2–5)]and F5008S [ISE, 4 (2–5); FP, 1 (−1–1.75); IC, 1 (−0.5 to 2)] mEq/L (P Conclusions We noted a systematic bias in DNa delivery with measured DNa being greater than that programmed by our HD machines. The magnitude of the bias varied between machines and with DNa. Our results may help explain the diverse results reported in studies of DNa.

Colicins are protein antibiotics deployed by Escherichia coli to eliminate competing strains. Colicins frequently exploit outer membrane (OM) nutrient transporters to penetrate the selectively permeable bacterial cell envelope. Here, by applying live-cell fluorescence imaging, we were able to monitor the entry of the pore-forming toxin colicin B (ColB) into E. coli and localize it within the periplasm. We further demonstrate that single-stranded DNA coupled to ColB can also be transported to the periplasm, emphasizing that the import routes of colicins can be exploited to carry large cargo molecules into bacteria. Moreover, we characterize the molecular mechanism of ColB association with its OM receptor FepA by applying a combination of photoactivated cross-linking, mass spectrometry, and structural modeling. We demonstrate that complex formation is coincident with large-scale conformational changes in the colicin. Thereafter, active transport of ColB through FepA involves the colicin taking the place of the N-terminal half of the plug domain that normally occludes this iron transporter. IMPORTANCE Decades of excessive use of readily available antibiotics has generated a global problem of antibiotic resistance and, hence, an urgent need for novel antibiotic solutions. Bacteriocins are protein-based antibiotics produced by bacteria to eliminate closely related competing bacterial strains. Bacteriocin toxins have evolved to bypass the complex cell envelope in order to kill bacterial cells. Here, we uncover the cellular penetration mechanism of a well-known but poorly understood bacteriocin called colicin B that is active against Escherichia coli. Moreover, we demonstrate that the colicin B-import pathway can be exploited to deliver conjugated DNA cargo into bacterial cells. Our work leads to a better understanding of the way bacteriocins, as potential alternative antibiotics, execute their mode of action as well as highlighting how they might even be exploited in the genomic manipulation of Gram-negative bacteria.

Cell penetrating peptides (CPPs) are short peptides that are able to translocate themselves and their cargo into cells. The progressive and continuous application of CPPs in various fields of basic and applied research shows that they are efficient delivery vectors for an assortment of biomolecules, including nucleic acids and proteins. This feature makes CPPs an excellent tool for modification of plant genomes through transgenesis and genome editing. In this review, we present the progress during the last three decades in application of CPPs for delivery of DNA, RNA, and proteins into plant cells and tissues. Moreover, we highlight the exploiting of CPPs as advantageous and beneficial tool for plant genome editing via delivery of nuclease proteins, and provide a practical example of genome alternation through CPP-delivered nucleases. Finally, the current exploitation of peptides in organelle-specific DNA delivery and modification of organellar genomes is discussed.

International audience; The use of cationic polymers in gene therapy aims at improving the circulation, protection and delivery of nucleic acids. The modification of poly(L-lysine) (PLL), including the insertion of hydrophobic moieties, is an efficient strategy to enhance the transfection efficiency of this synthetic delivery system. PLL is usually modified through either the extremities of the polymer chains or the amino groups present on the side chains. Herein the synthesis of dodecalysines as model of PLL with hexyl spacing units regularly distributed along the oligomer backbone is reported. Oligolysines terminated with azidohexyl groups were prepared by solid-phase synthesis and ligated by copper-assisted alkyne-azide cycloaddition using propargylamine. The iterative ligation of α-azidohexyloligolysines was more efficient when performed on resin as compared to the ligation in solution. The plasmid DNA transfection efficiency of the hydrophobically modified dodecalysines was then compared to that of unmodified dodecalysine. The results show that when formulated with the helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) the hydrophobized dodecalysines exhibited a higher efficiency than the unmodified one. Interestingly, the efficiency could also be increased by the addition of a fusogenic peptide, which paves the way for modifications of the oligolysines that could lead to improved activities.

This article provides a brief overview of DNA vaccines. First, the basic DNA vaccine design strategies are described, then specific issues related to the industrial production of DNA vaccines are discussed, including the production and purification of DNA products such as plasmid DNA, minicircle DNA, minimalistic, immunologically defined gene expression (MIDGE) and Doggybone™. The use of adjuvants to enhance the immunogenicity of DNA vaccines is then discussed. In addition, different delivery routes and several physical and chemical methods to increase the efficacy of DNA delivery into cells are explained. Recent preclinical and clinical trials of DNA vaccines for COVID-19 are then summarized. Lastly, the advantages and obstacles of DNA vaccines are discussed.

Inorganic cells bearing calcium silicate membranes were prepared and resembled closed chemical gardens. It was demonstrated that these inorganic cells can successfully be loaded with natural products, proteins and plasmid DNA, and their cargo can be released in a controlled manner. These cells demonstrated the ability of chemical gardens to act as platforms for the sustained delivery of biomolecules and are expected to introduce chemical gardens in the field of biosciences.

To noninvasively monitor the effect of gene therapy and achieve an optimal therapeutic effect, liposomes encapsulated polyethylenimine (PEI)-coated superparamagnetic iron oxide nanoparticles (SPION) with dual functions of MRI diagnosis and gene therapy were prepared. SPION was synthesized via co-precipitation, and then modified with PEI via thiourea reaction. The liposomes encapsulating PEI-SPION (LP-PEI-SPION) were prepared by ethanol injection. Fourier transform infrared spectra confirmed that PEI was successfully modified onto SPION, and thermogravimetric analysis indicated that the PEI content was about 17.1%. The LP-PEI-SPION/DNA had a small particle size of 253.07 ± 0.90 nm. LP-PEI-SPION/DNA had low cytotoxicity with more than 80% of the cell survival rates and high transfection efficiency compared with Lipofectamine® 2000/DNA. Additionally, it also showed good MRI effect on three cell lines. The liposomes encapsulating PEI-SPION (lipopolyplexes) have been successfully prepared as MRI contrast agents and gene delivery vectors, which may have great theoretical research significance and clinical potentials. Abbreviations: PEI, polyethylenimine; SPION, superparamagnetic iron oxide nanoparticles; LP-PEI-SPION, liposomes encapsulating PEI-SPION; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; ICP-MS, inductively coupled plasma mass spectrometry; XRD, X-ray diffraction; TEM, transmission electron microscope; TGA, thermogravimetric analysis; DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; Chol, cholesterol.

In continuation of previous work, we present a new promising DNA carrier, OO4, a highly effective peptide-mimicking lysine-based cationic lipid. The structural characteristics of the polynucleotide carrier system OO4 mixed with the commonly used co-lipid DOPE and the saturated phospholipid DPPE have been studied in two-dimensional and three-dimensional model systems to understand their influence on the physical–chemical properties. The phase behavior of pure OO4 and its mixtures with DOPE and DPPE was studied at the air–water interface using a Langmuir film balance combined with infrared reflection-absorption spectroscopy. In bulk, the self-assembling structures in the presence and absence of DNA were determined by small-angle and wide-angle X-ray scattering. The amount of adsorbed DNA to cationic lipid bilayers was measured using a quartz crystal microbalance. The choice of the co-lipid has an enormous influence on the structure and capability of binding DNA. DOPE promotes the formation of nonlamellar lipoplexes (cubic and hexagonal structures), whereas DPPE promotes the formation of lamellar lipoplexes. The correlation of the observed structures with the transfection efficiency and serum stability indicates that OO4/DOPE 1:3 lipoplexes with a DNA-containing cubic phase encapsulated in multilamellar structures seem to be most promising.

Chitosan-based particles are desirable materials for drug delivery because of their muco-adhesiveness in tissues, biocompatibility, low toxicity and effectiveness in antigen transport. It is still a challenge to prepare chitosan-based particles with high stability and by an effective method. Here we developed chitosan-based nanoparticles for the delivery of toll-like receptor 9 ligands, cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs), which can induce the change from Th-2 type to Th-1 type immune response. Because of the low tolerance to DNase and less uptake into the cells of the free CpG ODNs, the development of positively charged carriers is necessary for the effective delivery of CpG ODNs into immune cells. Chitosan nanoparticles were prepared by ionic gelation, and the nanoparticles size and surface charge were measured by dynamic light spectroscopy and zeta potential analyzer, respectively. The synthesized chitosan nanoparticles showed two dispersion peaks, at 196 ± 29 nm and 1.33 ± 0.36 μm, with a zeta potential of +3.3 ± 0.4 mV. The toxicity of the chitosan nanoparticles to murine RAW264 macrophages was measured by formazan dye assay utilizing the water-soluble tetrazolium salt WST-8. Our chitosan nanoparticles exhibited no cytotoxicity to RAW264 cells. Finally, we evaluated the immunostimulatory activity of CpG ODNs loaded on chitosan nanoparticles using human peripheral blood mononuclear cells. CpG ODNs significantly enhanced the secretion level of interleukin-6 and interferon-γ by cells compared to the free CpG ODNs. These results indicated that chitosan nanoparticles could be a good candidate for the delivery of CpG ODNs.

An effective delivery vehicle of genetic materials to their target site is the key to a successful gene therapy. In many cases, nanoparticles are used as the vehicle of choice and the efficiency of the delivery relies heavily on the physicochemical properties of the nanoparticles. Microfluidics, although being a low throughput method, has been increasingly researched for the preparation of nanoparticles. A range of superior properties were claimed in the literature for microfluidic-prepared platforms, but no evidence on direct comparison of the properties of the nanoparticles prepared by microfluidics and conventional high throughput method exists, leaving the industry with little guidance on how to select effective large-scale nanoparticle manufacturing method. This study used plasmid DNA-loaded PLGA-Eudragit nanoparticles as the model system to critically compare the nanoparticles prepared by conventional and microfluidics-assisted nanoprecipitation. The PLGA-Eudragit nanoparticles prepared by microfluidics were found to be statistically significantly larger than the ones prepared by conventional nanoprecipitation. PLGA-Eudragit nanoparticle prepared conventionally showed higher DNA loading efficiency. Although the DNA-loaded nanoparticles prepared by both methods did not induce significant cytotoxicity, the transfection efficiency was found to be higher for the ones prepared conventionally which has good potential for plasmid delivery. This study for the first time provides a direct comparison of the DNA-loaded nanoparticles prepared by microfluidic and conventional methods. The findings bring new insights into critical evaluation of the selection of manufacturing methods of nanoparticles for future gene therapy.

Chemotherapeutics represent the standard treatment for a wide range of cancers. However, these agents also affect healthy cells, thus leading to severe off-target effects. Given the non-selectivity of the commonly used drugs, any increase in the selective tumor tissue uptake would represent a significant improvement in cancer therapy. Recently, the use of gene therapy to completely remove the lesion and avoid the toxicity of chemotherapeutics has become a tendency in oncotherapy. Ideally, the genetic material must be safely transferred from the site of administration to the target cells, without involving healthy tissues. This can be achieved by encapsulating genes into non-viral carriers and modifying their surface with ligands with high selectivity and affinity for a relevant receptor on the target cells. Hence, in this work we evaluate the use of terpolymer-based nanocapsules for the targeted delivery of DNA toward cancer cells. The surface of the nanocapsules is decorated with folic acid to actively target the folate receptors overexpressed on a variety of cancer cells. The nanocapsules demonstrate a good ability of encapsulating and releasing DNA. Moreover, the presence of the targeting moieties on the surface of the nanocapsules favors cell uptake, opening up the possibility of more effective therapies.

The following protocol describes the genetic transformation of wheat using the BioRad PDS/1000-He particle delivery system. Immature embryos are isolated 12-16 days post anthesis, the embryonic axis is removed, and the immature scutella are pre-cultured for 1-2 days prior to particle bombardment. Gold particles are coated with plasmid DNA containing the gene(s) of interest plus a selectable marker gene, in this instance bar (bialaphos resistance), and are fired into the cells to deliver the DNA. Subsequent tissue culture and regeneration steps allow recovery of plantlets, assisted by the inclusion of PPT (phosphinothricin tripeptide), the active ingredient of glufosinate-ammonium containing herbicides, to help select transformants. This updated method introduces selection earlier in the regeneration process which provides a shortened protocol while maintaining high transformation efficiencies.

We recently developed a platform where phage-transducing particles optimize DNA delivery to a wide range of hosts. Here, we use this platform to optimize DNA transduction into hosts that naturally restrict specific DNA sequences. We first show that a specific plasmid is restricted for transduction into a particular Salmonella strain. Using the platform, we select for a mutated plasmid that overcomes the restriction barrier. Insertion of the non-mutated sequence into a permissive plasmid restricts transduction. We further show that epigenetic modification enables the DNA to evade restriction by the putative defense system. Our results validate this straightforward genetic approach for optimization of DNA transduction into new hosts.

The purpose of this paper is to prepare and evaluate a gene delivery system resulted from the intercalation of DNA with magnetic layered double hydroxide (MLDH). The structure and property of the MLDH/DNA hybrids were studied using XRD, FTIR and TG characterization. The cytotoxicity and protection effect of MLDH were evaluated by MTT assay and gel electrophoresis assay. MLDH exhibited lower cytotoxicity than other inorganic nanoparticles. Under physiological conditions, MLDH could protect DNA from enzymatic degradation. Our results confirmed that MLDH can be used safely and effectively as non-viral vectors for gene targeting therapy in form of MLDH/DNA composite.

Oct4 is a master regulator of the induction and maintenance of cellular pluripotency, and has crucial roles in early stages of differentiation. It is the only factor that cannot be substituted by other members of the same protein family to induce pluripotency. However, although Oct4 nuclear transport and delivery to target DNA are critical events for reprogramming to pluripotency, little is known about the molecular mechanism. Oct4 is imported to the nucleus by the classical nuclear transport mechanism, which requires importin α as an adaptor to bind the nuclear localization signal (NLS). Although there are structures of complexes of the NLS of transcription factors (TFs) in complex with importin α, there are no structures available for complexes involving intact TFs. We have therefore modeled the structure of the complex of the whole Oct4 POU domain and importin α2 using protein-protein docking and molecular dynamics. The model explains how the Ebola virus VP24 protein has a negative effect on the nuclear import of STAT1 by importin α but not on Oct4, and how Nup 50 facilitates cargo release from importin α. The model demonstrates the structural differences between the Oct4 importin α bound and DNA bound crystal states. We propose that the 'expanded linker' between the two DNA-binding domains of Oct4 is an intrinsically disordered region and that its conformational changes have a key role in the recognition/binding to both DNA and importin α. Moreover, we propose that this structural change enables efficient delivery to DNA after release from importin α.

The decoration of polyamidoamine (PAMAM) dendrimers with some functional moieties holds promises for improving the gene-delivery behavior of the resulting polyplexes. Herein, we reported the synthesis and the development of non-viral gene vectors built on an array of PAMAMs, spanning generation 2 (G2), G4, and G7, specifically tethered with multivalent neomycin (Neo) or guanidinoneomycin (GNeo). Following a thorough chemical characterization by means of NMR and MALDI analyses, the ability of PAMAM–(G)Neo conjugates to serve as transfectants was investigated. First, we demonstrated that every PAMAM, whether or not tethered with Neo (or GNeo), exhibited greater DNA packing ability than the gold standard transfectant 25 kDa bPEI. Measurements of the colloidal properties showed that PAMAMs, when mixed with plasmid DNA (pDNA) as a function of the nitrogen-to-phosphate ratio (N/P), allowed preparing complexes of ≈150–300 nm in diameter with a positive surface charge of ≈+20–35 mV, strictly dependent on the PAMA...

Cationic liposomes composed of 3-[N-(N&rsquo, N&rsquo, dimethylaminoethane)-carbamoyl] cholesterol (DC-chol) and dioleoylphosphatidylethanolamine (DOPE) have previously been shown to have applications in gene delivery. Our study aims to explore the effects of inclusion of polyethylene glycol (PEG) and using different molar ratios of DC-chol/DOPE on size, zeta potential, cytotoxicity and DNA delivery of DC-chol/DOPE liposomes. Our results show that PEGylation reduces the cytotoxicity of DC-chol/DOPE liposomes, and, furthermore, PEGylated liposome-DNA lipoplexes are smaller in size and more uniform in size distribution than those that are not PEGylated. Additionally, toxicity against ovarian cancer SKOV-3 cells decreases with the amount of cationic DC-chol present in the formulation, however, decreased delivery of DNA to cellular nuclei is also observed. Transfection with the PEGylated liposomes was successfully demonstrated using plasmid DNA with a known functional outcome. These results offer further insight into physicochemical properties important for cationic liposomes as vehicles for DNA delivery and demonstrate the potential of PEGylated DC-chol/DOPE liposomes as systemic delivery carriers for DNA-mediated ovarian cancer therapy.

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.

Mixtures of highly curved pyridinium-decorated Au nanoparticles and standard pyridinium cationic lipids efficiently and synergetically transfected DNA in vitro, while displaying good cytotoxic profile.

The microencapsulation process of bacteria has been used for many years, mainly in the food industry and, among the different matrixes used, sodium alginate stands out. This matrix forms a protective wall around the encapsulated bacterial culture, increasing its viability and protecting against environmental adversities, such as low pH, for example. The aim of the present study was to evaluate both in vitro and in vivo, the capacity of the encapsulation process to maintain viable lactic acid bacteria (LAB) strains for a longer period of time and to verify if they are able to reach further regions of mouse intestine. For this purpose, a recombinant strain of LAB (L. lactis ssp. cremoris MG1363) carrying the pExu vector encoding the fluorescence protein mCherry [L. lactis MG1363 (pExu:mCherry)] was constructed. The pExu was designed by our group and acts as a vector for DNA vaccines, enabling the host cell to produce the protein of interest. The functionality of the pExu:mCherry vector, was demonstrated in vitro by fluorescence microscopy and flow cytometry after transfection of eukaryotic cells. After this confirmation, the recombinant strain was submitted to encapsulation protocol with sodium alginate (1%). Non-encapsulated, as well as encapsulated strains were orally administered to C57BL/6 mice and the expression of mCherry protein was evaluated at different times (0-168 h) in different bowel portions. Confocal microscopy showed that the expression of mCherry was higher in animals who received the encapsulated strain in all portions of intestine analyzed. These results were confirmed by qRT-PCR assay. Therefore, this is the first study comparing encapsulated and non-encapsulated L. lactis bacteria for mucosal DNA delivery applications. Our results showed that the microencapsulation process is an effective method to improve DNA delivery, ensuring a greater number of viable bacteria are able to reach different sections of the bowel.

This paper describes the formulation of archaeosomes and the evaluation of their abilities to facilitate in vitro DNA delivery. Lipids of the H.hispanica 2TK2 strain were used in archaeosome formation, which is formulated by mixing H.hispanica 2TK2 lipids with plasmid DNA encoding green fluorescent protein (GFP) or β-galactosidase (β-gal). Archaeosome/pDNA formation and unbound DNA were monitored by agarose gel electrophoresis. The archaeosome formulations were visualized by AFM and TEM. The zeta potential analysis showed the archaeosomes to be electronegative. The composition of archaeosomes and the DNA dose for transient transfection into HEK293 cells were optimized, and the relationship between the structure and activity of archaeosomes in DNA delivery was investigated. By themselves, archaeosomes showed low efficiency for DNA delivery, due to their anionic nature. By formulating archaeosomes with a helper molecule, such as DOTAP, CaCl2, or LiCl, the capability of archaeosomes for gene transfection is significantly enhanced. The transfection profiles of efficient archaeosomes are proved to have a long shelf-life when maintained at room temperature. Thus, the archaeal lipids have the potential to be used as transfection reagents in vitro.

© 2017 American Chemical Society. After more than 20 years of intensive investigations, gene therapy has become one of the most promising strategies for treating genetic diseases. However, the lack of ideal delivery systems has limited the clinical realization of gene therapy's tremendous potential, especially for DNA-based gene therapy. Over the past decade, considerable advances have been made in the application of polymer-based DNA delivery systems for gene therapy, especially through multifunctional systems. The core concept behind multifunctional polymeric DNA delivery systems is to endow one single DNA carrier, via materials engineering and surface modification, with several active functions, e.g., good cargo DNA protection, excellent colloidal stability, high cellular uptake efficiency, efficient endo/lysosome escape, effective import into the nucleus, and DNA unpacking. Such specially developed vectors would be capable of overcoming multiple barriers to the successful delivery of DNA. In this review, we first provide a comprehensive overview of the interactions between the protein corona and DNA vectors, the mechanisms and challenges of nonviral DNA vectors, and important concepts in the design of DNA carriers identified via past reports on DNA delivery systems. Finally, we highlight and discuss recent advances in multifunctional polymeric DNA delivery systems based on "off-the-shelf" polycations including polyethylenimine (PEI), poly-l-lysine (PLL), and chitosan and offer perspectives on future developments.

International audience; Lactic acid bacteria as Lactococcus lactis are used as vector for protein but also DNA delivery into intestinal cells in vitro and in vivo. For the plasmid delivery strategy, the plasmid copy number per bacteria (PCN) is thus of great importance. The aim of this paper is to determine the physiological conditions when PCN is the highest in the bacteria. PCN was characterized by qPCR in five different recombinant Lactococcus lactis strains, containing one (mono-) or two different plasmids (biplasmidic), at exponential or stationary phase. We showed that in all cases but one, PCN is higher at exponential than stationary phase. PCN seems to depend on (i) monoplasmidic or biplasmidic strain; (ii) origin of replication of the plasmid; and (iii) the DNA load of the bacteria. Then we studied plasmid transfer in vitro from recombinant L. lactis to eukaryotic COS-7 cells using culture at exponential or stationary phase. We showed that plasmid transfer can be improved in vitro by using bacteria at exponential phase.

An unnatural CDP-amino acid (kd) is used in the design and synthesis of novel cell penetrating peptidomimetic (Kkd-5). The balanced cationic nature and structural rigidity of Kkd-5 resulted in serum stability, non-toxicity to cells, and interaction with membrane and DNA, all of which facilitated efficient cellular uptake and DNA delivery.

The success of nonviral gene therapy mainly depends on the design and development of safe and effective gene delivery systems. Nonviral vectors have attracted the attention of researchers, as they offer several advantages over their viral counterparts. Among various nonviral vectors, polyethylenimines have been the most widely used in in vitro and in vivo gene transfer studies. Cationic nanoparticles have been shown to enhance the efficiency of the transfection. These are compact and help in overcoming various biological and cellular barriers. Due to their smaller size, these are less susceptible to reticuloendothelial (RES) clearance and exhibit better uptake and internalization into the cells/tissues. To exploit these properties of the nanoparticles, the present chapter highlights the synthesis of nanoparticles of polyethylenimine (linear and branched) and their ability to effect gene transfer across the biological systems.

There is much progress in application of genetic engineering for improving the biological properties of different organisms. Viral and nonviral carriers are used for delivery of genetic material into target cells. Polymeric materials of natural and synthetic origin are the most promising gene delivery agents. These polymers demonstrated high efficiency of DNA delivery into animal cells, although they were not very effective in plant cells. Here, the procedure for genetic transformation of Ceratodon purpureus (Hedw.) Brid. moss protoplasts is described. The method is based on the application of surface-active polymeric carriers of the poly-DMAEM structure and controlled length and charge. This allows obtaining more transient and stable moss transformants per microgram of plasmid DNA when compared with known protocol based on using polyethyleneglycol. It is easier, more convenient, and cheaper than the “gene gun” method. Prospects for further improvement of structure and functional characteristics of new polymeric carriers are considered for delivery of genetic material into plant cells.

The emergence of photoluminescent carbon-based nanomaterials offers great potential for a wide variety of biomedical applications such as fluorescence imaging and cellular labeling. This report illustrates a novel photonic carbon dot (Cdots) based nanocarrier by using low molecular weight amphiphilic PEI (Alkyl-PEI2k) for surface passivation. The resulting water-dispersible Alkyl-PEI2k-Cdot nanocarrier possesses good stability, monodispersity with narrow size distribution and fluorescence properties. In addition, Alkyl-PEI2k-Cdot nanocarrier has a markedly low toxicity and good gene transfection effect in vitro and in vivo. Considering its low cytotoxicity, high gene delivery efficiency and fluorescence performance, Alkyl-PEI2k-Cdots could serve as a novel imaging-trackable gene-delivery nanocarrier promising for gene therapy and optical molecular imaging.

Polyamidoamine (PAMAM) dendrimers are emerging as intriguing nanovectors for nucleic acid delivery because of their unique well-defined architecture and high binding capacity, which have been broadly applied in DNA- and RNA-based therapeutics. The low-cost and high-efficiency of PAMAM dendrimers relative to traditional liposomal transfection reagents also promote their application in gene function analysis. In this study, we first investigated the potential use of a PAMAM system in the silkworm model insect. We determined the binding property of G5-PAMAM using dsRNA and DNA in vitro, and substantially achieved the delivery of dsRNA and DNA from culture medium to both silkworm BmN and BmE cells, thus leading to efficient knockdown and expression of target genes. Under treatments with different concentrations of G5-PAMAM, we evaluated its cellular cytotoxicity on silkworm cells, and the results show that G5-PAMAM had no obvious toxicity to cells. The presence of serum in the culture medium did not affect the delivery performance of DNA and dsRNA by G5-PAMAM, revealing its convenient use for various purposes. In conclusion, our data demonstrate that the PAMAM system provides a promising strategy for delivering dsRNA and DNA in cultured silkworm cells and promote its further application in individuals.

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.

Magnetic gold nanoparticles (mGNPs) with uniform size and morphology synthesized by our sonication treatment method were covalently bound with fluorescein isothiocyanate (FITC) molecules. Driven by an external magnetic field, FITC-labelled nanoparticles were delivered into plant cells with and without cell walls, evident from sectional transmission electron microscopy images. Confocal images further indicate that the green fluorescence in canola protoplasts and walled cells indeed came from the FITC molecules, instead of the chloroplasts’ autofluorescence. FITC-labelled nanoparticles had a delivery efficiency of 95% based on confocal images. In further study, plasmids were covalently bound with mGNPs, and delivered into canola cells with and without cell walls. After culturing for 48 h followed by staining with 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid (X-Gluc), blue colour appeared in the protoplasts, while the walled canola cells showed a green colour that can be interpreted as the combination of blue and yellow from the suspension cells themselves. The presence of the blue colour indicates the expression of the GUS gene; therefore, the plasmids were successfully delivered into the canola cells. Furthermore, on examination, mGNPs were considered to be noncytotoxic by fluorescein diacetate staining.

Conjugation of episomal plasmids from bacteria to diatoms advances diatom genetic manipulation by simplifying transgene delivery and providing a stable and consistent gene expression platform. To reach its full potential, this nascent technology requires new optimized expression vectors and a deeper understanding of episome maintenance. Here, we present the development of an additional diatom vector (pPtPBR1), based on the parent plasmid pBR322, to add a plasmid maintained at medium copy number in Escherichia coli to the diatom genetic toolkit. Using this new vector, we evaluated the contribution of individual yeast DNA elements comprising the 1.4-kb tripartite CEN6-ARSH4-HIS3 sequence that enables episome maintenance in Phaeodactylum tricornutum. While various combinations of these individual elements enable efficient conjugation and high exconjugant yield in P. tricornutum, individual elements alone do not. Conjugation of episomes containing CEN6-ARSH4 and a small sequence from the low GC content 3' end of HIS3 produced the highest number of diatom exconjugant colonies, resulting in a smaller and more efficient vector design. Our findings suggest that the CEN6 and ARSH4 sequences function differently in yeast and diatoms, and that low GC content regions of greater than ~500 bp are a potential indicator of a functional diatom episome maintenance sequence. Additionally, we have developed improvements to the conjugation protocol including a high-throughput option utilizing 12-well plates and plating methods that improve exconjugant yield and reduce time and materials required for the conjugation protocol. The data presented offer additional information regarding the mechanism by which the yeast-derived sequence enables diatom episome maintenance and demonstrate options for flexible vector design.

Gene electrotransfer is a powerful method of DNA delivery offering several medical applications, among the most promising of which are DNA vaccination and gene therapy for cancer treatment. Electroporation entails the application of electric fields to cells which then experience a local and transient change of membrane permeability. Although gene electrotransfer has been extensively studied in in vitro and in vivo environments, the mechanisms by which DNA enters and navigates through cells are not fully understood. Here we present a comprehensive review of the body of knowledge concerning gene electrotransfer that has been accumulated over the last three decades. For that purpose, after briefly reviewing the medical applications that gene electrotransfer can provide, we outline membrane electropermeabilization, a key process for the delivery of DNA and smaller molecules. Since gene electrotransfer is a multipart process, we proceed our review in describing step by step our current understanding, with particular emphasis on DNA internalization and intracellular trafficking. Finally, we turn our attention to in vivo testing and methodology for gene electrotransfer.

Jin Sil Lee, Won Hur Department of Bioengineering and Technology, Kangwon National University, Chuncheon, South Korea Abstract: Purified fibroin protein can be obtained in large quantities from silk fibers and processed to form microscopic particles as delivery vehicles for therapeutic agents. In this study, we demonstrated that fibroin microspheres were taken up by 3T3 cells, localized in the nonlysosomal compartment, and secreted from the cytoplasm after medium replenishment. DNA-loaded microspheres were taken up by >95% of 3T3 cells. DNA cargo had no influence on the intracellular trafficking of microspheres, while fluorescently labeled cargo DNA was observed in the lysosomal compartment and in the microspheres. These results indicate that fibroin microspheres can travel through 3T3 cells without making any contact with the lysosomal compartments. The amount of DNA loaded in the microspheres taken up by 3T3 cells was estimated up to 831.0pg/cell. Thus, fibroin microspheres can deliver a large amount of randomly fragmented DNA (

Electroporation, an attractive process for delivering DNA and other molecules into target cells in vivo and in vitro is limited by the necessity of electrodes that need to be in contact with the subject or object to be electroporated. We have used magnetic fields, which do not require material contact with the subject, to temporarily permeabilize cells in guinea pig skin in vivo to enhance uptake and expression of GFP plasmid DNA. The results show for the first time that magnetic fields can trigger a process likely similar to electroporation. In designing the magnetic pulses, our most important criterion was a high rate of change of the magnetic field, based on the principle described by Michael Faraday which is expressed by the formula: E = -dB/dt, (E, electric field, B, magnetic field, t, time). Magnetic fields were generated by a flat electromagnet in a hand-held applicator positioned above the target tissue. The magnetic pulses had a peak magnetic flux density of 4 tesla; 50 pulses were applied in 5 sec. Biphasic magnetic pulses were twice as effective as monophasic pulses and about equally effective as traditional electroporation pulses . Advantages of magnetopermeabilization over electoporation include: No contact between applicator and subject (“contact-less”); no need for invasive, disposable, sterile electrodes (“needle-less”); no pain from needles and reduced overall pain; no known side effects; easier and faster to administer than electroporation; less expensive due to absence of disposables; and, importantly, greater tissue penetration of the magnetic field allowing treatment of anatomical areas inaccessible by electroporation.

Polycations are a promising class of nonviral DNA delivery agents that bind to negatively charged DNA and transfect the DNA into target cells. The architecture and chemistry of the polycation strongly affect polycation–DNA complexation and in turn the ability of polycations to transfect DNA into cells. Here we develop coarse-grained models and conduct Langevin dynamics simulations to understand how the architecture of lysine-based polycations affects their complexation with DNA-like polyanions. We first characterize the structure of linear polylysine and oligolysines grafted to a polyolefin backbone and then the structure of complexes (termed polyplexes) formed by these polycations with polyanions of varying flexibility. We find that increasing oligolysine graft length and decreasing graft spacing both increase the size and rigidity of the grafted oligolysines, although they remain less rigid than semiflexible linear polylysine. Increasing ionic strength or counterion valency reduces polycation size and m...

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.

Xun Sun, Simu Chen, Jianfeng Han, Zhirong ZhangKey Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, People’s Republic of ChinaBackground: To establish a potential gene-delivery system with the ability to deliver plasmid DNA to dendritic cells (DCs) more efficiently and specifically, we designed and synthesized a low-molecular-weight polyethyleneimine and triethyleneglycol polymer (PEI–TEG) and a series of its mannosylated derivatives.Methods: PEI–TEG was synthesized from PEI2000 and PEI600 with TEG as the cross-linker. PEI–TEG was then linked to mannose via a phenylisothiocyanate bridge to obtain man-PEI–TEG conjugates. The DNA conveyance abilities of PEI–TEG, man-PEI–TEG, as well as control PEI25k were evaluated by measuring their zeta potential, particle size, and DNA-binding abilities. The in vitro cytotoxicity, cell uptake, and transfection efficiency of these PEI/DNA complexes were examined on the DC2.4 cell line. Finally, a maturation experiment evaluated the effect of costimulatory molecules CD40, CD80, and CD86 on murine bone marrow-derived DCs (BMDCs) using flow cytometry.Results: PEI–TEG and man-PEI–TEG were successfully synthesized and were shown to retain the excellent properties of PEI25k for condensing DNA. Compared with PEI–TEG as well as PEI25k, the man-PEI–TEG had less cytotoxicity and performed better in both cellular uptake and transfection assays in vitro. The results of the maturation experiment showed that all the PEI/DNA complexes induced an adequate upregulation of surface markers for DC maturation.Conclusion: These results demonstrated that man-PEI–TEG can be employed as a DC-targeting gene-delivery system.Keywords: dendritic cells, DCs, mannose, polyethyleneimine, PEI, gene delivery

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.

Gene, short hairpin RNA (shRNA) and small interfering RNA (siRNA) delivery can be particularly used for the treatment of diseases by the entry of genetic materials mammalian cells either to express new proteins or to suppress the expression of proteins, respectively. Polyamidoamine (PAMAM) StarburstTM dendrimers are used as non-viral vectors (carriers) for gene, shRNA and siRNA delivery. Recently, multifunctional PAMAM dendrimers can be used for the wide range of biomedical applications including intracellular delivery of genes and nucleic acid drugs. In this context, this review paper provides the recent findings on PAMAM dendrimer conjugates with cyclodextrins (CyDs) for gene, shRNA and siRNA delivery.

Gene therapy offers greater opportunities for treating numerous incurable diseases from genetic disorders, infections, and cancer. However, development of appropriate delivery systems could be one of the most important factors to overcome numerous biological barriers for delivery of various therapeutic molecules. A number of nonviral polymer-mediated vectors have been developed for DNA delivery and offer the potential to surmount the associated problems of their viral counterpart. To address the concerns associated with safety issues, a wide range of polymeric vectors are available and have been utilized successfully to deliver their therapeutics in vivo. Today's research is mainly focused on the various natural or synthetic polymer-based delivery carriers that protect the DNA molecule from degradation, which offer specific targeting to the desired cells after systemic administration, have transfection efficiencies equivalent to virus-mediated gene delivery, and have long-term gene expression through sustained-release mechanisms. This review explores an updated overview of different nonviral polymeric delivery system for delivery of DNA-based therapeutics. These polymeric carriers have been evaluated in vitro and in vivo and are being utilized in various stages of clinical evaluation. Continued research and understanding of the principles of polymer-based gene delivery systems will enable us to develop new and efficient delivery systems for the delivery of DNA-based therapeutics to achieve the goal of efficacious and specific gene therapy for a vast array of clinical disorders as the therapeutic solutions of tomorrow.

A combinatorial library of lipidoids was constructed and studied for in vitro gene delivery. The library of lipidoids was synthesized by reacting commercially available amines with lipophilic acrylates, acrylamides, or epoxides. Lipidoids derived from amine 86 (N,N-Bis(2-hydroxyethyl)ethylene diamine) and amine 87 (N-(3-aminopropyl)diethaneamine) showed high efficiency in DNA delivery, some with a higher transfection efficiency than Lipofectamine 2000, a commonly used commercial gold standard for in vitro gene delivery. The structure-activity relationship between the lipidoids was further studied with respect to small variations in chemical structures and the resulting efficiency in DNA delivery in vitro. Since these lipidoids are easy to synthesize and do not require a co-lipid for efficient DNA delivery, they could offer an inexpensive but effective alternative to other commonly used commercial gene delivery carriers.

Novel DNA-chitosan particles were prepared based on associative phase separation and interfacial diffusion. These particles formed at water/water emulsion type interfaces were characterized with respect to several properties including stability, DNA conformational state, and entrapment and release of DNA. In particular it was found that the chitosan molecular weight is a good controlling parameter.