DNA Delivery Systems Research Articles

Ion milling coupled field emission scanning electron microscopy reveals current misunderstanding of morphology of polymeric nanoparticles

Nanoparticles (NPs) are currently used as drug delivery systems for numerous therapeutic macromolecules, e.g. proteins or DNA. Based on the preparation by double emulsion solvent evaporation a sponge-like structure was postulated entrapping hydrophilic drugs inside an internal aqueous phase. However, a direct proof of this hypothesized structure is still missing today. NPs were prepared from different polymers using a double-emulsion method and characterized for their physicochemical properties. Combining ion milling with field emission scanning electron microscopy allowed to cross section single NP and to visualize their internal morphology. The imaging procedure permitted cross-sectioning of NPs and visualization of the internal structure as well as localizing drugs associated with NPs. It was observed that none of the model actives was encapsulated inside the polymeric matrix when particle diameters were below around 470nm but predominantly adsorbed to the particle surface. Even at larger diameters only a minority of particles of a diameter below 1μm contained an internal phase. The properties of such drug loaded NPs, i.e. drug release or the observations in cellular uptake or even drug targeting needs to be interpreted carefully since in most cases NP surface properties are potentially dominated by the ‘encapsulated’ drug characteristics.

Immunogenicity of a DNA and Recombinant Protein Vaccine Combining LipL32 and Loa22 for Leptospirosis Using Chitosan as a Delivery System.

Leptospirosis is a worldwide zoonotic disease caused by pathogenic Leptospira, a genus of which more than 250 serovars have been identified. Commercial bacterin vaccines are limited in that they lack both cross-protection against heterologous serovars and long-term protection. This study investigated in mice the immunogenicity of an anti-leptospirosis vaccine, using the outer membrane proteins LipL32 and Loa22 as antigens. The immunogenicity of this vaccine formulation was compared with those induced by vaccines based on LipL32 or Loa22 alone. A DNA-encapsulated chitosan nanoparticle was used for in vivo DNA delivery. Using a unique DNA plasmid expressing both lipL32 and loa22 for vaccination, higher antibody responses were induced than when combining plasmids harboring each gene separately. Therefore, this formulation was used to test the immunogenicity when administered by a heterologous prime (DNA)-boost (protein) immunization regimen. The specific antibody responses against LipL32 (total IgG and IgG1) and Loa22 (IgG1) were higher in mice receiving two antigens in combination than in those vaccinated with a single antigen alone. Although no significant difference in splenic CD4+ T cell proliferation was observed among all groups of vaccinated mice, splenocytes from mice vaccinated with two antigens exhibited higher interferon-γ and IL-2 production than when using single antigens alone upon in vitro restimulation. Taken together, the immunogenicity induced by LipL32 and Loa22 antigens in a heterologous primeboost immunization regimen using chitosan as a DNA delivery system induces higher immune response, and may be useful for developing a better vaccine for leptospirosis.

Selective DNA delivery to tumor cells using an oligoarginine-LTVSPWY peptide.

DNA therapy for cancer requires efficient, selective and safe DNA delivery systems. Compared with other non-viral methods such as lipid or polymer-based DNA delivery vectors, peptide-based DNA delivery systems are biocompatible and biodegradable, which leads to lower immunogenicity and lower toxicity. Moreover, peptide vectors are easier to produce and their compositions easier to control because solid-phase peptide synthesis has been extensively developed. However, peptide-based systems for DNA delivery toward special tumor cells or tissues are still lacking. In this study, we constructed a non-viral 9rR-LTVSPWY peptide-based DNA delivery system and showed that it is able to efficiently and selectively transfect DNA into targeted tumor cells. This work presents a novel strategy for tumor cell-specific DNA delivery and a reference for designing more efficient DNA delivery systems targeted towards various types of cancer.

DMAEM-based cationic polymers as novel carriers for DNA delivery into cells.

Different transformation systems and vectors have been improved to increase the effectiveness of transformation and achieve stable expression of target genes. Because classical direct and indirect transformation processes commonly suffer from instability of a gene in the environment, gene deletion, transgene silencing, and poor gene transfer efficiency. Nowadays, gene transformation technologies are based on the use of new carriers (nanoparticles, carbon nanotubes, whiskers, and polymers) characterized by better efficiency and reproducibility for the direct DNA delivery into cells. In this review, we have focused on the novel DMAEM-based direct DNA delivery system and its possible applications for cell transformation.

Chestnut, American (Castanea dentata (Marsh.) Borkh.).

The key to successful transformation of American chestnut is having the correct combination of explant tissue, selectable markers, a very robust DNA delivery system, and a reliable regeneration system. The most important components of this transformation protocol for American chestnut are the following: starting out with rapidly dividing somatic embryos, treating the embryos gently throughout the Agrobacterium inoculation and cocultivation steps, doing the cocultivation step in desiccation plates, and finally transferring the embryos into temporary-immersion bioreactors for selection. None of these departures from standard Agrobacterium transformation protocols is sufficient by itself to achieve transgenic American chestnut, but each component makes a difference, resulting in a highly robust protocol. The average transformation efficiency that can be expected using the described protocol is approximately 170 stable embryogenic transformation events per gram of somatic embryo tissue, a considerable improvement over the 20 transformation events per gram we reported in 2006 (Maynard et al. American chestnut (Castanea dentata (Marsh.) Borkh.) Agrobacterium protocols, 2nd ed., 2006). We have regenerated nearly 100 of these events, containing 23 different gene constructs, into whole plants. As of the fall of 2013, we had a total of 1,275 transgenic chestnut trees planted at eight locations in New York State and one in Virginia. Based on a combination of field-trial inoculations, greenhouse small-stem inoculations, and detached-leaf assays, we have identified three transgenes that produce stronger resistance to chestnut blight than non-transgenic American chestnut. Depending on the transgene and the event, this resistance can be either intermediate between American chestnut and Chinese chestnut, approximately equal to or even higher than the resistance naturally found in Chinese chestnut.

Tc52 amino-terminal-domain DNA carried by attenuated Salmonella enterica serovar Typhimurium induces protection against a Trypanosoma cruzi lethal challenge.

In this work we immunized mice with DNA encoding full-length Tc52 or its amino- or carboxy-terminal (N- and C-term, respectively) domain carried by attenuated Salmonella as a DNA delivery system. As expected, Salmonella-mediated DNA delivery resulted in low antibody titers and a predominantly Th1 response, as shown by the ratio of IgG2a/IgG1-specific antibodies. Despite modest expression of Tc52 in trypomastigotes, the antibodies elicited by vaccination were able to mediate lysis of the trypomastigotes in the presence of complement and inhibit their invasion of mammal cells in vitro. The strongest functional activity was observed with sera from mice immunized with Salmonella carrying the N-term domain (SN-term), followed by Tc52 (STc52), and the C-term domain (SC-term). All immunized groups developed strong cellular responses, with predominant activation of Th1 cells. However, mice immunized with SN-term showed higher levels of interleukin-10 (IL-10), counterbalancing the inflammatory reaction, and also strong activation of Tc52-specific gamma interferon-positive (IFN-γ(+)) CD8(+) T cells. In agreement with this, although all prototypes conferred protection against infection, immunization with SN-term promoted greater protection than that with SC-term for all parameters tested and slightly better protection than that with STc52, especially in the acute stage of infection. We conclude that the N-terminal domain of Tc52 is the section of the protein that confers maximal protection against infection and propose it as a promising candidate for vaccine development.

Sandwich-type Au-PEI/DNA/PEI-Dexa nanocomplex for nucleus-targeted gene delivery in vitro and in vivo.

Many synthetic Au-based cationic nanoparticles (AuNPs) for nonviral gene delivery show high efficiency in vitro, but their excessive charge density, harsh reducing conditions, and nontarget delivery prevent their application in vivo. Herein, we constructed a sandwich-type layered polyethylenimine (PEI)-coated gold nanocomposite outerlaid with a nucleus-targeted Dexamethasone (Dexa), namely, Au-PEI/DNA/PEI-Dexa nanocomplex, for DNA delivery system using a low molecular weight PEI as a mild reducing agent. The nucleus-targeting Au-PEI/DNA/PEI-Dexa nanocomplex with low positive charge and low cytotoxicity condensed DNA and protected from enzymatic degradation. In vitro transfection studies demonstrated that Au-PEI/DNA/PEI-Dexa nanocomplex exhibited much more efficient nucleus transfection than Au-PEI/DNA/PEI without nucleus-targeted residues and commercially available PEI 25 kDa due to the Dexa targeting of the nucleus. Furthermore, the nanocomplex markedly transfected pTRAIL (TRAIL = tumor-necrosis-factor-related apoptosis-inducing ligand) to tumors in vivo and subsequently inhibited the tumor growth with minimal side effects. These findings suggest that nucleus-targeting Au-PEI/DNA/PEI-Dexa ternary complexes have promising potential in gene delivery.

A novel in vitro system for intracellular delivery of nonviral DNA

Articular chondrocytes are the main cell population in cartilage, and damage to them is a key step in osteoarthritis. There are chondrocyte-based strategies to treat osteoarthritis, among which includes nonviral gene therapy. However, so far there is no ideal way to achieve this because chondrocyte cells are very difficult to transfect. Also, an effective tracking system to evaluate exogenous DNA delivery in chondrocytes is a necessary part of this strategy. Here, we show our development of a novel tracking system by labelling cell membranes, nuclei, and plasmids, without disturbing their expression, to view the intracellular behaviour of plasmids before, after, and during the entire transfection process. We applied this system to compare the intracellular behaviour of exogenous DNA in chondrocytes and cancer cells. We also used this system to compare the intracellular behaviour of exogenous DNA which is transfected by liposomes or polymers. Finally, we discovered that when transfected by liposomes, exogenous DNA has a quicker cell entry and nucleus entry in cancer cells than in chondrocytes, and the transfection efficiency is higher in cancer cells than in chondrocytes. When the cells are transfected by polymers, exogenous DNA has the same quick cell entry and nucleus entry in both types of cells. However, the transfection efficiency was higher in cancer cells than in chondrocytes. Applying this system has proved to be simple, easy to operate, repeatable, and stable and it enables tracking of the behaviour of plasmid DNA before and after expression. Meanwhile, the rate-limiting zones of exogenous plasmid DNA in living cells and a comprehensive overview of the expression and transfection efficiency of DNA can also be obtained through this system, making it ideal for the research and development of nonviral DNA delivery systems.

Efficient targeted gene delivery by a novel PAMAM/DNA dendriplex coated with hyaluronic acid.

To design and develop a novel target-specific DNA-delivery system using hyaluronic acid (HA)-polyamidoamine (PAMAM) conjugates (P-HA). The coupling of HA to the PAMAM dendrimer was analyzed by (1)H-NMR and elemental analysis (CHN). Their properties were characterized in terms of size and zeta-potential and evaluated for in vitro and in vivo transfection efficiency. The designed covalent HA-dendriplexes enhanced gene transfection of pCMV-Luc reporter gene in overexpressing CD44-receptor cancer cells. They were also more efficient in transfecting MDA-MB231 cells than conventional PEI-polyplexes. The cytotoxicity of the covalent HA-dendriplexes was lower than when using conventional polyethylenimine-polyplexes. In vivo studies showed that these targeted complexes were also efficient for delivering pCMVLuc in different organs of healthy mice, as well as in tumors of C57BL/6 animals. The HA-dendriplexes developed in this work may offer an advantageous alternative to conventional cationic polymer-based formulations for DNA delivery into cancer cells in an efficient and safe manner.

Development of functional polyplex micelles for systemic gene therapy

Recent advances in chemistry-based engineering have developed smart nanodevices for realizing targeted therapy by rationally integrating versatile molecular technologies to achieve specific structures and functionalities. Gene therapy is one of the most intriguing targets, because nanodevices to deliver fragile DNA into pathological cells and exert effective transgene expression are indispensable for achieving this innovative therapeutic modality. The largest challenge lies in the development of an appropriate systemic delivery system that can withstand the harsh environment of circulating blood and efficiently accumulate in a targeted location for gene expression. This review focuses mainly on our polymer-based DNA delivery systems, which are fabricated from polyion complexation between plasmid DNA and functionalized poly(ethylene glycol)-based block catiomers, and highlights their development to systemic settings for application to cancer treatment. Smart polyplex micelles for systemic gene therapy fabricated by rationally integrating versatile molecular-based technologies. The polyplex micelles, which are formed through an electrostatic interaction-mediated self-assembly process of functional block copolymers and plasmid DNA, assume a task to transport therapeutic gene into nucleus of pathological cells via systemic route for functional protein expression.

Optimisation of Synthetic Vector Systems for Cancer Gene Therapy – The Role of the Excess of Cationic Dendrimer Under Physiological Conditions

We have previously demonstrated in a therapeutic study that a single systemic course of DAB-Am16 dendriplexes loaded with plasmid expressing TNFα over a period of time of 10 days led to a regression of 100% of tumours and to long term cures of up to 80% of animals. However, the formulation had a relatively low colloidal stability requiring administration soon after nanoparticle preparation. Similar to other cationic polyplex and dendrimer DNA delivery systems, DAB-AM16 dendrimer formulations contained a substantial proportion of free polymer; this free polymer is present independently of the specific polymer:DNA ratio and increases with increasing proportion of polymer (N:P charge ratio) in the formulation. It has previously been shown for this and other systems that the excess of polymer plays a role in promoting the transfection efficiency of synthetic vectors. This has been linked to effects of the polymer on the efficiency of intracellular processing, e.g. endosomal release. However, the free polymer may have additional effects that are relevant to the efficiency of the formulation. This study therefore considered the effect of free dendrimer on the colloidal stability of the complexes, the interaction of the complex with the formulation medium, and with biological components, i.e. electrolytes and serum proteins after administration. Analysis of the total potential of interaction shows that, even at high N:P ratios, the excess of free dendrimer in the medium is not enough to induce the aggregation of the formulation due to depletion forces. This finding is unusual and can be attributed to the particularly low Mw of these dendrimers (1.6 kDa). On the other hand, formulations are highly sensitive to the strength of the dendrimer:DNA interactions. These can be controlled by the degree of protonation (α) of the dendrimer which is strongly dependent on bulk pH. Modulation of the protonation level to α≥0.4 allows reproducible production of colloidally stable particles. Finally, we have demonstrated that electrolytes and proteins present in physiological media play a crucial role to favour the efficiency of these synthetic vectors reducing the toxicity associated with their cationic groups.

Gene therapy and DNA delivery systems

Gene therapy is a promising new technique for treating many serious incurable diseases, such as cancer and genetic disorders. The main problem limiting the application of this strategy in vivo is the difficulty of transporting large, fragile and negatively charged molecules like DNA into the nucleus of the cell without degradation. The key to success of gene therapy is to create safe and efficient gene delivery vehicles. Ideally, the vehicle must be able to remain in the bloodstream for a long time and avoid uptake by the mononuclear phagocyte system, in order to ensure its arrival at the desired targets. Moreover, this carrier must also be able to transport the DNA efficiently into the cell cytoplasm, avoiding lysosomal degradation. Viral vehicles are the most commonly used carriers for delivering DNA and have long been used for their high efficiency. However, these vehicles can trigger dangerous immunological responses. Scientists need to find safer and cheaper alternatives. Consequently, the non-viral carriers are being prepared and developed until techniques for encapsulating DNA can be found. This review highlights gene therapy as a new promising technique used to treat many incurable diseases and the different strategies used to transfer DNA, taking into account that introducing DNA into the cell nucleus without degradation is essential for the success of this therapeutic technique.

High-Throughput Methods for Screening Polymeric Transfection Reagents

The clinical success of gene therapy requires the development of a safe and efficient delivery system for DNA. Cationic polymers are nonviral vectors that can associate electrostatically with plasmid DNA to form nanocomplexes. In some cases, this is sufficient for cellular uptake and transfection, although the precise mechanisms by which polymers facilitate gene delivery remain unclear. A robust and reliable method to screen for efficacy is essential for the development of effective polycationic transfection reagents. Numerous parameters must be controlled and optimized, such as polymer structure, polymer-DNA-binding conditions (mixing method, pH, ionic strength, incubation time, concentrations, ratios), transfection media (type, serum content), DNA dose and incubation time with the cells, cell specificity, and assay conditions. In this protocol, we describe a high-throughput method for assessing polymer-mediated transfection. The technique uses 96-well plates, which allows many transfection parameters to be varied and optimized in parallel. Hundreds of polymers can be tested in quadruplicate in a single day and the technique can easily be automated to efficiently and reproducibly test large material libraries. One limitation is that many plate types, solutions, and equipment must be stocked and sterilized. Moreover, because all polymers are processed simultaneously in very small volumes, it is difficult to validate each step for each polymer to ensure solution uniformity and adequate polymer-DNA complexation. Despite these drawbacks, this high-throughput screening method has already been used successfully in the development of efficient polycation vectors.

Interfacial engineering of pyridinium gemini surfactants for the generation of synthetic transfection systems

Pyridinium gemini surfactants possess a soft charge, a high charge/mass ratio and a high molecular flexibility – all key parameters that recommend their use in synthetic gene delivery systems with in vitro and in vivo efficiency. In present study we generated a DNA delivery system through interfacial engineering of pyridinium gemini surfactants at the level of linker, hydrophobic chains and counterions. The self-assembling of the pyridinium amphiphiles and the physicochemical properties of the resultant supra-molecular assemblies were studied in bulk and in solution through a combination of techniques that included DSC, X-ray diffraction, polarized microscopy, CMC, dynamic light scattering and zeta potential measurements. We assessed the impact of different structural elements and formulation parameters of these pyridinium amphiphiles on their DNA compaction properties, transfection efficiency, cytotoxicity, in a complete structure–activity relationship study. This interfacial engineering process generated transfection systems with reduced cytotoxicity and high transfection efficiency in media containing elevated levels of serum that mimic the in vivo conditions.

Systemically administered gp100 encoding DNA vaccine for melanoma using water-in-oil-in-water multiple emulsion delivery systems

The purpose of this study was to develop a water-in-oil-in-water (W/O/W) multiple emulsions-based vaccine delivery system for plasmid DNA encoding the gp100 peptide antigen for melanoma immunotherapy. The gp100 encoding plasmid DNA was encapsulated in the inner-most aqueous phase of squalane oil containing W/O/W multiple emulsions using a two-step emulsification method. In vitro transfection ability of the encapsulated plasmid DNA was investigated in murine dendritic cells by transgene expression analysis using fluorescence microscopy and ELISA methods. Prophylactic immunization using the W/O/W multiple emulsions encapsulated the gp100 encoding plasmid DNA vaccine significantly reduced tumor volume in C57BL/6 mice during subsequent B16-F10 tumor challenge. In addition, serum Th1 cytokine levels and immuno-histochemistry of excised tumor tissues indicated activation of cytotoxic T-lymphocytes mediated anti-tumor immunity causing tumor growth suppression. The W/O/W multiple emulsions-based vaccine delivery system efficiently delivers the gp100 plasmid DNA to induce cell-mediated anti-tumor immunity.

Network analysis of endogenous gene expression profiles after polyethyleneimine‐mediated DNA delivery

DNA delivery systems, which transport exogenous DNA to cells, have applications that include gene therapy, tissue engineering and medical devices. Although the cationic nonviral DNA carrier polyethyleneimine (PEI) has been widely studied, the molecular factors and pathways underlying PEI-mediated DNA transfer remain largely unknown, preventing the design of more efficient delivery systems. HEK 293 T cells were treated with polyplexes formed with PEI and pEGFPLuc encoding for green fluorescent protein (GFP). Transfected cells expressing GFP were flow-separated from treated, untransfected cells. Gene expression profiles were obtained using Affymetrix HG-U133 2.0 microarrays and differentially expressed genes were identified using R/Bioconductor. Gene network analysis using EGAN (exploratory gene association network) bioinformatics tools was then used to find interaction among genes and enriched gene ontology (GO) terms related to transfection. Genes identified by this method were perturbed using pharmacologic activators or inhibitors to assess their effect on DNA transfer. Microarray analysis comparing transfected cells to untransfected cells revealed 215 genes to be differentially expressed, with the majority enriched to GO processes including metabolism, response to stimulus, cell cycle, biological regulation and cellular component organization or biogenesis pathways. Gene network analysis revealed a coordinated induction of RAP1A, SCG5, PGAP1, ATF3 and NEB genes implicated in cell stress, cell cycle and cytoskeletal processes. Altering pathways with pharmacologic agents confirmed the potential role of RAP1A, SCG5 and ATF3 in transfection. Microarray and gene network analyses of the sorted, transfected cell population can identify potential mediators of transfection, providing a basis for the design of improved delivery systems.

Freeze Dried Chitosan/ Poly-ε-Caprolactone and Poly-ε-Caprolactone Nanoparticles: Evaluation of their Potential as DNA and Antigen Delivery Systems

Nanoparticles prepared from natural or synthetic polymers have shown potential for antigen and DNA vaccine delivery to mucosal surfaces. The purpose of this research was to prepare chitosan/poly-e-caprolactone (Chitosan/ PCL) nanoparticles and PCL nanoparticles and evaluate their potential as DNA and protein/vaccine delivery systems. Both preparation methods resulted in particles of low cytotoxicity and sizes suggested to be ideal to be taken up by cells (199 ± 62 nm and 165 ± 35 nm, respectively for chitosan/PCL and PCL nanoparticles). However, Chitosan/ PCL nanoparticles offered considerable advantages over PCL nanoparticles as antigen and DNA delivery system. Namely, higher loading efficacies for model antigens studied (myoglobin, BSA, ovalbumin, lactalbumin, α-casein and lysozyme), much higher uptake by A549 cells, great ability to form stable complexes, which protect DNA from nucleases. However, in spite of good DNA and protein loading capabilities, Chitosan/PCL nanoparticles showed much better qualities as a protein delivery system since the rate of cells transfected were not very high.

Chitosan conjugates for DNA delivery

Graft copolymers of chitosan and maltodextrin were synthesized by reductive amination of a low molecular weight chitosan. The degree of substitution is 70% or above, as quantified by nuclear magnetic resonance spectroscopy, at molar ratios of chitosan glucosamine units and maltodextrin of 1 : 1 and 1 : 5. The high substitution degree generates a water-soluble compound at pH 7.4 by reducing the amino groups of chitosan. In addition, the copolymer self-assembles into spherical nanoparticles with diameters of about 300 nm. The mass polymer/DNA ratios at the isoelectric point are within the range of 3-3.5 for chitosan-maltodextrin nanoparticles as determined by electrophoretic mobility measurements. These results confirm that DNA interacts efficiently with the copolymer nanoparticles indicating a potential application of the system for DNA delivery.

A gene delivery method mediated by three arginine-rich cell-penetrating peptides in plant cells

The transient gene delivery systems are generally mediated by viral infection, particle bombardment, electroporation, and microinjection for transferring exogenous DNAs into host cells. Recently, the peptide-mediated DNA delivery system became a novel tool for gene transfer, and these peptides, such as cell-penetrating peptides (CPPs), contained the ability of permeating plasma membranes and carrying cargoes to enter cells. In this study, we use three different arginine-rich CPPs, synthetic nona-arginine (SR9), histidine-rich nona-arginine (HR9), and Pas nona-arginine (PR9), for gene delivery into root-tip cells of mung bean and rice. We demonstrate that these arginine-rich CPPs have the ability to transfer Cyanine 3 (Cy3)-labeled DNAs into plant cells. Moreover, the CPP-mediated DNA transfer in cells was confirmed by polymerase chain reaction (PCR) analysis. Finally, our results of the functional gene assay indicated that DNA exerts its biological activity after intracellular delivery via CPPs. This CPP-mediated gene delivery method is characterized by the simplicity, noninvasion, and efficiency. Three arginine-rich CPPs may be ideal tools for the research of plant gene transformation.

DNA Delivery into Mammalian Cells Using Bacteriophage λ Displaying the TAT Transduction Domain

The ability to modulate cellular function by the transfer and expression of novel genes or by affecting the levels of endogenous proteins by genetic means has been of tremendous benefit in studying cellular functions and offers great promise in the treatment of a variety of diseases. Consequently, the development of novel and efficient nonviral DNA delivery systems is an important goal. Small cationic peptides, termed peptide/protein transduction domains (PTDs), effectively deliver a wide variety of cargoes, including DNA, into all cells. Expression of the human immunodeficiency virus type 1 (HIV-1) TAT PTD on the surface of bacteriophage λ results in the efficient delivery of plasmid DNA into a variety of cells in a concentration-dependent manner without cytotoxicity. This protocol describes the preparation of recombinant λ particles with a TAT peptide transduction domain sequence on their surface and the use of these particles in delivery of plasmid DNA into a variety of cells.