E. coli mediated gene delivery faces a major drawback of low efficiency despite of being a safer alternative to viral vectors. This study showed a novel, simple and effective strategy to enhance invasive E. coli DH10B vector's efficiency in human epithelial cells. The bactofection efficiency of invasive E .coli vector was analyzed in nine cell lines. It demonstrated highest (16%) reporter gene (GFP) expression in cervical cells. Methods were employed to further enhance its efficiency by adding transfection reagents (trans-bactofection method) to promote entry into host cells, lysosomotropic reagents for escape from lysosomal degradation or antibiotics to lyse internalized bacteria. Increased bacterial entry, as elucidated from nil to 3% expression in liver cells, was obtained upon complexing bacteria with PULSin. Chloroquine mediated endosomal escape resulted in 7.2 folds increase whereas tetracycline addition to lyse internalized bacteria caused ≈90% of GFP in HeLa. Eventually, the combined effect of these three methods exhibited close to 100% GFP in cervical and remarkable increase of 138 folds in breast cells. This is the first study showing comparative study of vector's gene delivery ability in various epithelial cells of the human body with improving its delivery efficiency. These data demonstrated the potential of developed bactofection method to boost up the efficiency of other bacterial vectors also, which could further be used for effectual therapeutic gene delivery in human cells.
Aptamers, nucleic acid ligands that are specific against their corresponding targets are increasingly employed in a variety of applications including diagnostics and therapeutics. The specificity of the aptamers against their targets is also used as the basis for the formulation of the aptamer-based drug delivery system. In this review, we aim to provide an overview on the chaperoning roles of aptamers in acting as the cargo or load carriers, delivering contents to the targeted sites via cell surface receptors. Internalization of the aptamer-biomolecule conjugates via receptor-mediated endocytosis and the strategies to augment the rate of endocytosis are underscored. The cargos chaperoned by aptamers, ranging from siRNAs to DNA origami are illuminated. Possible impediments to the aptamer-based drug deliveries such as susceptibility to nuclease resistance, potentiality for immunogenicity activation, tumor heterogeneity are speculated and the corresponding amendment strategies to address these shortcomings are discussed. We prophesy that the future of the aptamer-based drug delivery will take a trajectory towards DNA nanorobot-based assay.
Gene therapy research has advanced to clinical trials, but it is hampered by unstable nucleic acids packaged inside carriers and there is a lack of specificity towards targeted sites in the body. This study aims to address gene therapy limitations by encapsidating a plasmid synthesizing a short hairpin RNA (shRNA) that targets the anti-apoptotic Bcl-2 gene using truncated hepatitis B core antigen (tHBcAg) virus-like particle (VLP). A shRNA sequence targeting anti-apoptotic Bcl-2 was synthesized and cloned into the pSilencer 2.0-U6 vector. The recombinant plasmid, namely PshRNA, was encapsidated inside tHBcAg VLP and conjugated with folic acid (FA) to produce FA-tHBcAg-PshRNA VLP. Electron microscopy revealed that the FA-tHBcAg-PshRNA VLP has an icosahedral structure that is similar to the unmodified tHBcAg VLP. Delivery of FA-tHBcAg-PshRNA VLP into HeLa cells overexpressing the folate receptor significantly downregulated the expression of anti-apoptotic Bcl-2 at 48 and 72 h post-transfection. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay demonstrated that the cells' viability was significantly reduced from 89.46% at 24 h to 64.52% and 60.63%, respectively, at 48 and 72 h post-transfection. As a conclusion, tHBcAg VLP can be used as a carrier for a receptor-mediated targeted delivery of a therapeutic plasmid encoding shRNA for gene silencing in cancer cells.
The application of nanoparticles (NPs) has attracted considerable attention as targeted delivery systems. CaCO3 has become the focus due to its advantages including affordability, low toxicity, biocompatibility, cytocompatibility, pH sensitivity and sedate biodegradability and environment friendly materials. In this article, we will discuss the po- tential roles of CaCO3-NPs in three major therapeutic applications; as antimicrobial, for drug delivery, and as gene delivery nanocarrier.
Vascular endothelial growth factor (VEGF) is a potent angiogenic factor involved in angiogenesis-mediated progression of acute myeloid leukemia (AML). Studies have reported the role of soluble form of fms-like tyrosine kinase (sFlT-1) delivery as an antitumor agent by inhibiting VEGF. This study investigates the outcome of delivery of a VEGF165 antagonist, soluble vascular endothelial growth factor receptor, namely sFLT-1, mediating lipofectamine 2000 in acute myeloid leukemic cells. A recombinant plasmid expressing sFLT-1 was constructed and transfected into the K562 and HL60 cells using lipofectamine 2000 transfection reagent. sFLT-1 expression/secretion in pVAX-sFLT-1 transfected cells was verified by RT-PCR and western blot. MTS assay was carried out to evaluate the effect of sFLT-1 on human umbilical vein endothelial cells and K562 and HL60 cells in vitro. Treatment with pVAX-sFLT-1 showed no association between sFLT-1 and proliferation of infected K562 and HL60 cells, while it demonstrated a significant inhibitory impact on the proliferation of HUVECs. The results of the current study imply that the combination of nonviral gene carrier and sFLT-1 possesses the potential to provide efficient tool for the antiangiogenic gene therapy of AML.
Leukemic cells are hard-to-transfect cell lines. Many transfection reagents which can provide high gene transfer efficiency in common adherent cell lines are not effective to transfect established blood cell lines or primary leukemic cells. This study aims to examine a new class of cationic polymer non-viral vector, PEGylated-dextran-spermine (PEG-D-SPM), to determine its ability to transfect the leukemic cells. Here, the optimal conditions of the complex preparation (PEG-D-SPM/plasmid DNA (pDNA)) were examined. Different weight-mixing (w/w) ratios of PEG-D-SPM/pDNA complex were prepared to obtain an ideal mixing ratio to protect encapsulated pDNA from DNase degradation and to determine the optimal transfection efficiency of the complex. Strong complexation between polymer and pDNA in agarose gel electrophoresis and protection of pDNA from DNase were detected at ratios from 25 to 15. Highest gene expression was detected at w/w ratio of 18 in HL60 and K562 cells. However, gene expression from both leukemic cell lines was lower than the control MCF-7 cells. The cytotoxicity of PEG-D-SPM/pDNA complex at the most optimal mixing ratios was tested in HL60 and K562 cells using MTS assay and the results showed that the PEG-D-SPM/pDNA complex had no cytotoxic effect on these cell lines. Spherical shape and nano-nature of PEG-D-SPM/pDNA complex at ratio 18 was observed using transmission electron microscopy. As PEG-D-SPM showed modest transfection efficiency in the leukemic cell lines, we conclude that further work is needed to improve the delivery efficiency of the PEG-D-SPM.
Alginate, a natural polysaccharide, was explored in this study as an oral delivery vehicle of a mammalian expression vector into the murine intestinal mucosa. Alginate microspheres were produced through water-in-oil (W/O) emulsification method. Average diameter sizes of microspheres were 46.88 μm±3.07 μm with significant size reduction upon utilization of 1.0% Span80. Plasmid DNA (pDNA) carrying green fluorescent protein reporter gene (GFP), pVAX-GFP, was encapsulated within microspheres at efficiencies of 72.9 to 74.4%, carrying maximum load of 6 μg pDNA. Alginate microspheres demonstrated shrinkage in pH 1.2 and swelling in pH 9.0 with pDNA release about twice the amount released in acidic environment. Oral delivery of pVAX-GFP loaded-microspheres, at 50 μg, 100 μg and 150 μg dose, was performed on BALB/c mice. Tissue biodistribution, investigated through flow cytometric analysis, demonstrated GFP positive intestinal cells (<1.0%) with 1.3-fold higher levels for the 100 μg dose; therefore suggesting feasibility of the approach for oral gene delivery and vaccination.
Cellulose acetate phthalate (CAP) microcapsules were formulated to deliver plasmid DNA (pDNA) to the intestines. The microcapsules were characterized and were found to have an average diameter of 44.33 ± 30.22 μm, and were observed to be spherical with smooth surface. The method to extract pDNA from CAP was modified to study the release profile of the pDNA. The encapsulated pDNA was found to be stable. Exposure to the acidic and basic pH conditions, which simulates the pH environment in the stomach and the intestines, showed that the release occurred in a stable manner in the former, whereas it was robust in the latter. The loading capacity and encapsulation efficiency of the microcapsules were low but the CAP recovery yield was high which indicates that the microcapsules were efficiently formed but the loading of pDNA can be improved. In vitro transfection study in 293FT cells showed that there was a significant percentage of green-fluorescent-protein-positive cells as a result of efficient transfection from CAP-encapsulated pDNA. Biodistribution studies in BALB/c mice indicate that DNA was released at the stomach and intestinal regions. CAP microcapsules loaded with pDNA, as described in this study, may be useful for potential gene delivery to the intestines for prophylactic or therapeutic measures for gastrointestinal diseases.
Morinda citrifolia, is a valuable medicinal plant with a wide range of therapeutic properties and extensive transformation study on this plant has yet been known. Present study was conducted to establish a simple and reliable transformation protocol for M. citrifolia utilising Agrobacterium tumefaciens via direct seed exposure. In this study, the seeds were processed by tips clipping and dried and subsequently incubated in inoculation medium. Four different parameters during the incubation such as incubation period, bacterial density, temperature and binary vectors harbouring beta-glucuronidase (GUS) gene (pBI121 and pGSA1131), were tested to examine its effect on transformation efficiency. The leaves from the treated and germinated seedlings were analysed via Polymerase Chain Reaction (PCR), histochemical assay of the GUS gene and reverse transcription-PCR (RT-PCR). Results of the study showed that Agrobacterium strain LBA4404 with optical density of 1.0 and 2 h incubation period were optimum for M. citrifolia transformation. It was found that various co-cultivation temperatures tested and type of vector used did not affect the transformation efficiency. The highest transformation efficiency for M. citrifolia direct seed transformation harbouring pBI121 and pGSA1131 was determined to be 96.8% with 2 h co-cultivation treatment and 80.4% when using bacterial density of 1.0, respectively. The transformation method can be applied for future characterization study of M. citrifolia.
Polyethylenimine (PEI) is one of the most broadly used polycations for gene delivery due to its high transfection efficiency and commercial availability but materials are cytotoxic and often polydisperse. The goal of current work is to develop an alternative family of polycations based on controlled living radical polymerization (CLRP) and to optimize the polymer structure for efficient gene delivery. In this study, well-defined poly(glycidyl methacrylate)(P(GMA)) homopolymers were synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization followed by decoration using three different types of oligoamines, i.e., tetraethylenepentamine (TEPA), pentaethylenehexamine (PEHA), and tris(2-aminoethyl)amine (TREN), respectively, to generate various P(GMA-oligoamine) homopolycations. The effect of P(GMA) backbone length and structure of oligoamine on gene transfer efficiency was then determined. The optimal polymer, P(GMA-TEPA)(50), provided comparable transfection efficiency but lower cytotoxicity than PEI. P(GMA-TEPA)(50) was then used as the cationic block in diblock copolymers containing hydrophilic N-(2-hydroxypropyl) methacrylamide (HPMA) and oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA). Polyplexes of block copolymers were stable against aggregation in physiological salt condition and in Opti-MEM due to the shielding effect of P(HPMA) and P(OEGMA). However, the presence of the HPMA/OEGMA block significantly decreased the transfection efficacy of P(GMA-TEPA)(50) homopolycation. To compensate for reduced cell uptake caused by the hydrophilic shell of polyplex, the integrin-binding peptide, RGD, was conjugated to the hydrophilic chain end of P(OEGMA)(15)-b-P(GMA-TEPA)(50) copolymer by Michael-type addition reaction. At low polymer to DNA ratios, the RGD-functionalized polymer showed increased gene delivery efficiency to HeLa cells compared to analogous polymers lacking RGD.
Development of in vitro plant regeneration method from Brassica explants via organogenesis and somatic embryogenesis is influenced by many factors such as culture environment, culture medium composition, explant sources, and genotypes which are reviewed in this study. An efficient in vitro regeneration system to allow genetic transformation of Brassica is a crucial tool for improving its economical value. Methods to optimize transformation protocols for the efficient introduction of desirable traits, and a comparative analysis of these methods are also reviewed. Hence, binary vectors, selectable marker genes, minimum inhibitory concentration of selection agents, reporter marker genes, preculture media, Agrobacterium concentration and regeneration ability of putative transformants for improvement of Agrobacterium-mediated transformation of Brassica are discussed.
PURPOSE: In order to enhance cellular uptake and to facilitate transdermal delivery of DNA vaccine, polyamidoamine (PAMAM) dendrimers conjugated with HIV transactivator of transcription (TAT) was developed.
METHODS: First, the plasmid DNA (pIRES-H5/GFP) nanoparticle was formulated using PAMAM dendrimer and TAT peptide and then characterized for surface charge, particle size, DNA encapsulation and protection of the pIRES-H5/GFP DNA plasmid to enzymatic digestion. Subsequently, the potency of the TAT-conjugated dendrimer for gene delivery was evaluated through in vitro transfection into Vero cells followed by gene expression analysis including western blotting, fluorescent microscopy and PCR. The effect of the TAT peptide on cellular uptake of DNA vaccine was studied by qRT-PCR and flow cytometry. Finally, the ability of TAT-conjugated PAMAM dendrimer for transdermal delivery of the DNA plasmid was assessed through artificial membranes followed by qRT-PCR and flow cytometry.
RESULTS: TAT-conjugated PAMAM dendrimer showed the ability to form a compact and nanometre-sized polyplexes with the plasmid DNA, having the size range of 105 to 115 nm and a positive charge of +42 to +45 mV over the N/P ratio of 6:1(+/-). In vitro transfection analysis into Vero cells confirms the high potency of TAT-conjugated PAMAM dendrimer to enhance the cellular uptake of DNA vaccine. The permeability value assay through artificial membranes reveals that TAT-conjugated PAMAM has more capacity for transdermal delivery of the DNA compared to unmodified PAMAM dendrimer (P<0.05).
CONCLUSIONS: The findings of this study suggest that TAT-conjugated PAMAM dendrimer is a promising non-viral vector for transdermal use.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.
Direct protein delivery into the cytosol of mammalian cells by invasive Escherichia coli (E. coli) bacterial vector will bypass the need to achieve nuclear entry and transcription of DNA, a major hurdle that is known to seriously limit gene transfer. The bacterial vector is induced to express the protein during its growth phase, before presentation for entry into mammalian cells and release of its content into the cellular environment. For this class of vector, crossing the plasma membrane becomes the primary step that determines the success of protein delivery. Yet, how the mechanics of protein expression within the vector affect its entry into the host is poorly understood. We found the vector's effectiveness to enter HeLa cells diminished together with its viability when phage N15 protelomerase (TelN) expression was induced continuously in the invasive E. coli despite producing an abundant amount of functional protein. By comparison, shorter induction, even as little as 3 hr, produced sufficient amounts of functional TelN and showed more effective invasion of HeLa cells, comparable to that of uninduced invasive E. coli. These results demonstrate that brief induction of protein expression during vector growth is essential for optimal entry into mammalian cells, an important step for achieving bacteria-mediated protein delivery.
Gene delivery using invasive bacteria as vectors is a robust method that is feasible for plasmid and artificial chromosome DNA construct delivery to human cells presenting β1 integrin receptors. This technique is relatively underutilized owing to the inefficiency of gene transfer to targeted cell populations. Bacterial vectors must successfully adhere to the cell membrane, internalize into the cytoplasm, undergo lysis, and deliver DNA to the nucleus. There are limited studies on the use of exogenous reagents to improve the efficiency of bacteria-mediated gene delivery to mammalian cells. In this chapter, we describe how cationic lipids, conventionally used for DNA and protein transfection, as well as antimicrobial compounds, can be used to synergistically enhance the adherence of invasive bacterial vectors to the cell membrane and improve their predisposition to internalize into the cytoplasm to deliver DNA. Using simple combinatorial methods, functional DNA transfer can be improved by up to four-fold of invaded cell populations. These methods are easy to perform and are likely to be applicable for other bacterial vectors including Listeria and Salmonella.
Visual analysis of the gene delivery process when using invasive bacteria as a vector has been conventionally performed using standard light and fluorescence microscopy. These microscopes can provide basic information on the invasiveness of the bacterial vector including the ability of the vector to successfully adhere to the cell membrane. Standard microscopy techniques however fall short when finer details including membrane attachment as well as internalization into the cytoplasm are desired. High-resolution visual analysis of bacteria-mediated gene delivery can allow accurate measurement of the adherence and internalization capabilities of engineered vectors. Here, we describe the use of scanning electron microscopy (SEM) to directly quantify vectors when they are external to the cell wall, and confocal microscopy to evaluate the vectors when they have internalized into the cytoplasm. By performing the invasion procedure on microscope coverslips, cells can be easily prepared for analysis using electron or confocal microscopes. Imaging the invasion complexes in high resolution can provide important insights into the behavior of bacterial vectors including E. coli, Listeria, and Salmonella when invading their target cells to deliver DNA and other molecules.
Sample preparation for scanning electron microscope analysis involves reagents and equipment that are expensive and often hazardous. Here we demonstrate a circumvention of Osmium tetroxide and critical point drying, greatly reducing the duration, complexity and cost of the process. We captured early stage interactions of invasive-bacteria and HeLa cells during the process of bacteria-mediated gene delivery and illustrate sufficient clarity can be obtained using this procedure to preserve and clearly visualize relevant cellular structures. This protocol is significantly cheaper and easier to adapt compared to conventional methods, and will allow routine preparation/viewing of eukaryotic or bacterial samples for basic morphological studies.
Commonly used visible markers for transgenesis use fluorescent proteins expressed at the surface of the body, such as in eyes. One commonly used marker is the 3xP3-EGFP cassette containing synthetic binding sites for the eyeless/Pax6 conserved transcription factor. This marker cassette leads to fluorescent eyes in a variety of animals tested so far. Here we show that upon reaching adulthood, transgenic Bicyclus anynana butterflies containing this marker cassette exponentially loose fluorescence in their eyes. After 12 days, transgenic individuals are no longer distinguishable from wild type individuals. The decreased eye fluorescence is likely due to significantly decreased or halted eyeless/Pax6 expression observed in wild type animals upon adult emergence. Implications from these findings include care in screening transgenic animals before these reach adulthood, or shortly thereafter, and in using adult animals of the same age for quantitative screening of likely homozygote and heterozygote individuals.
Nanotechnology has provided new technological opportunities, which could help in challenges confronting stem cell research. Polyamidoamine (PAMAM) dendrimers, a new class of macromolecular polymers with high molecular uniformity, narrow molecular distribution specific size and shape and highly functionalised terminal surface have been extensively explored for biomedical application. PAMAM dendrimers are also nanospherical, hyperbranched and monodispersive molecules exhibiting exclusive properties which make them potential carriers for drug and gene delivery.
The possibility of controlling Fusarium wilt--caused by Fusarium oxysporum sp. cubensec (race 4)--was investigated by genetic engineering of banana plants for constitutive expression of rice thaumatin-like protein (tlp) gene. Transgene was introduced to cauliflower-like bodies' cluster, induced from meristemic parts of male inflorescences, using particle bombardment with plasmid carrying a rice tlp gene driving by the CaMV 35S promoter. Hygromycin B was used as the selection reagent. The presence and integration of rice tlp gene in genomic DNA confirmed by PCR and Southern blot analyses. RT-PCR revealed the expression of transgene in leaf and root tissues in transformants. Bioassay of transgenic banana plants challenged with Fusarium wilt pathogen showed that expression of TLP enhanced resistance to F. oxysporum sp. cubensec (race 4) compared to control plants.
Rice is one of the most important cereal crops with great potential for biotechnology progress. In transformation method, antibiotic resistance genes are routinely used as powerful markers for selecting transformed cells from surrounding non-transformed cells. In this study, the toxicity level of hygromycin was optimized for two selected mutant rice lines, MR219 line 4 and line 9. The mature embryos were isolated and cultured on an MS medium with different hygromycin concentrations (0, 20, 40, 60, 80 and 100 mg L(-1)). Evidently, above 60 mg L(-1) was effective for callus formation and observed completely dead. Further there were tested for specific concentration (0-60). Although, 21.28% calli survived on the medium containing 45 mg L(-1) hygromycin, it seemed suitable for the identification of putative transformants. These findings indicated that a system for rice transformation in a relatively high frequency and the transgenes are stably expressed in the transgenic plants. Green shoots were regenerated from the explant under hygromycin stress. RT-PCR using hptII and gus sequence specific primer and Southern blot analysis were used to confirm the presence of the transgene and to determine the transformation efficiency for their stable integration in regenerated plants. This study demonstrated that the hygromycin resistance can be used as an effective marker for rice transformation.