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Despite the great progress over the past few decades in both the diagnosis and treatment of a great variety of human cancers, glioblastoma remains the most lethal brain tumor. In recent years, cancer gene therapy focused on non-viral vectors which emerged as a promising approach to glioblastoma treatment. Transferrin (Tf) easily penetrates brain cells of the blood–brain barrier, and its receptor is highly expressed in this barrier and glioblastoma cells. Therefore, the development of delivery systems containing Tf appears as a reliable strategy to improve their brain cells targeting ability and cellular uptake. In this work, a cell-penetrating peptide (WRAP5), bearing a Tf-targeting sequence, has been exploited to condense tumor suppressor p53-encoding plasmid DNA (pDNA) for the development of nanocomplexes. To increase the functionality of developed nanocomplexes, the drug Temozolomide (TMZ) was also incorporated into the formulations. The physicochemical properties of peptide/pDNA complexes were revealed to be dependent on the nitrogen to phosphate groups ratio and can be optimized to promote efficient cellular internalization. A confocal microscopy study showed the capacity of developed complexes for efficient glioblastoma cell transfection and consequent pDNA delivery into the nucleus, where efficient gene expression took place, followed by p53 protein production. Of promise, these peptide/pDNA complexes induced a significant decrease in the viability of glioblastoma cells. The set of data reported significantly support further in vitro research to evaluate the therapeutic potential of developed complexes against glioblastoma. [ABSTRACT FROM AUTHOR]

Objectives: Epitope-driven vaccines carrying highly conserved and immunodominant epitopes have emerged as promising approaches to overcome human immunodeficiency virus-1 (HIV-1) infection. Methods: Two multiepitope DNA constructs encoding T cell epitopes from HIV-1 Gag, Pol, Env, Nef and Rev proteins alone and/or linked to the immunogenic epitopes derived from heat shock protein 70 (Hsp70) as an immunostimulatory agent were designed. In silico analyses were applied including MHC-I and MHC-II binding, MHC-I immunogenicity and antigen processing, population coverage, conservancy, allergenicity, toxicity and hemotoxicity. The peptide-MHC-I/MHC-II molecular docking and cytokine production analyses were carried out for predicted epitopes. The selected highly immunogenic T-cell epitopes were then used to design two multiepitope fusion constructs. Next, prediction of the physicochemical and structural properties, B cell epitopes, and constructs-toll-like receptors (TLRs) molecular docking were performed for each construct. Finally, the eukaryotic expression plasmids harboring totally 12 cytotoxic T Lymphocyte (CTL) and 10 helper T lymphocytes (HTL) epitopes from HIV-1 proteins (i.e., pEGFP-N1-gag-pol-env-nef-rev), and linked to 2 CTL and 2 HTL epitopes from Hsp70 (i.e., pEGFP-N1-hsp70-gag-pol-env-nef-rev) were generated and transfected into HEK-293 T cells for evaluating the percentage of multiepitope peptides expression using flow cytometry and western blotting. Results: The designed DNA constructs could be successfully expressed in mammalian cells. The expression rates of Gag-Pol-Env-Nef-Rev-GFP and Hsp70-Gag-Pol-Env-Nef-Rev-GFP were about 56–60% as the bands of ~ 63 and ~ 72 kDa confirmed in western blotting, respectively. Conclusion: The combined in silico/in vitro methods indicated two multiepitope constructs can be produced and used as probable effective immunogens for HIV-1 vaccine development. [ABSTRACT FROM AUTHOR]

Despite the great progress over the past few decades in both the diagnosis and treatment of a great variety of human cancers, glioblastoma remains the most lethal brain tumor. In recent years, cancer gene therapy focused on non-viral vectors which emerged as a promising approach to glioblastoma treatment. Transferrin (Tf) easily penetrates brain cells of the blood–brain barrier, and its receptor is highly expressed in this barrier and glioblastoma cells. Therefore, the development of delivery systems containing Tf appears as a reliable strategy to improve their brain cells targeting ability and cellular uptake. In this work, a cell-penetrating peptide (WRAP5), bearing a Tf-targeting sequence, has been exploited to condense tumor suppressor p53-encoding plasmid DNA (pDNA) for the development of nanocomplexes. To increase the functionality of developed nanocomplexes, the drug Temozolomide (TMZ) was also incorporated into the formulations. The physicochemical properties of peptide/pDNA complexes were revealed to be dependent on the nitrogen to phosphate groups ratio and can be optimized to promote efficient cellular internalization. A confocal microscopy study showed the capacity of developed complexes for efficient glioblastoma cell transfection and consequent pDNA delivery into the nucleus, where efficient gene expression took place, followed by p53 protein production. Of promise, these peptide/pDNA complexes induced a significant decrease in the viability of glioblastoma cells. The set of data reported significantly support further in vitro research to evaluate the therapeutic potential of developed complexes against glioblastoma.

Transfection by means of non-viral gene delivery vectors is the cornerstone of modern gene delivery. Despite the resources poured into the development of ever more effective transfectants, improvement is still slow and limited. Of note, the performance of any gene delivery vector in vitro is strictly dependent on several experimental conditions specific to each laboratory. The lack of standard tests has thus largely contributed to the flood of inconsistent data underpinning the reproducibility crisis. A way researchers seek to address this issue is by gauging the effectiveness of newly synthesized gene delivery vectors with respect to benchmarks of seemingly well-known behavior. However, the performance of such reference molecules is also affected by the testing conditions. This survey points to non-standardized transfection settings and limited information on variables deemed relevant in this context as the major cause of such misalignments. This review provides a catalog of conditions optimized for the gold standard and internal reference, 25 kDa polyethyleneimine, that can be profitably replicated across studies for the sake of comparison. Overall, we wish to pave the way for the implementation of standardized protocols in order to make the evaluation of the effectiveness of transfectants as unbiased as possible.

Efficient gene delivery facilitated by non-viral vectors, is comparatively safer alternative than viral carriers. Current approaches to gene delivery largely depends on methods that overcome cellular and tissue barriers impeding efficient DNA delivery. While the conventional delivery systems have the drawback of low cellular uptake and off target effects, the receptor-mediated gene delivery system has shown remarkable breakthrough. In that context exploiting the specific receptor targeting properties of lactoferrin protein, we have developed pDNA loaded lactoferrin protein nanoparticles (pDNA-LfNPs). pDNA-LfNPs were spherical in shape within the size range of 140 ± 20 nm. They were found to be resistant against nuclease digestion and stable under long storage. Additionally, LfNPs were biocompatible and have targeting ability to facilitate gene uptake by receptor mediated internalization in lactoferrin receptor expressing cell lines. It is also evident from our studies that Lf nanoparticles did not exhibit any cytotoxicity even at the highest concentration. Here we first time report the use of lactoferrin nanoparticles as a gene delivery carrier with targeting abilities. [ABSTRACT FROM AUTHOR]

Protein expression and RNA interference require efficient delivery of DNA or mRNA and small double stranded RNA into cells, respectively. Although cationic lipids are the most commonly used synthetic delivery vectors, a clear need still exists for a better delivery of various types of nucleic acids molecules to improve their biological activity. To optimize the transfection efficiency, a molecular approach consisting in modifying the chemical structure of a given cationic lipid is usually performed, but an alternative strategy could rely on modulating the supramolecular assembly of lipidic lamellar phases sandwiching the nucleic acids molecules. To validate this new concept, we synthesized on one hand two paromomycin-based cationic lipids, with either an amide or a phosphoramide linker, and on the other hand two imidazole-based neutral lipids, having as well either an amide or a phosphoramide function as linker. Combinations of cationic and helper lipids containing the same amide or phosphoramide linkers led to the formation of homogeneous lamellar phases, while hybrid lamellar phases were obtained when the linkers on the cationic and helper lipids were different. Cryo-transmission electron microscopy and fluorescence experiments showed that liposomes/nucleic acids complexes resulting from the association of nucleic acids with hybrid lamellar phases led to complexes that were more stable in the extracellular compartment compared to those obtained with homogeneous systems. In addition, we observed that the most active supramolecular assemblies for the delivery of DNA, mRNA and siRNA were obtained when the cationic and helper lipids possess linkers of different natures. The results clearly show that this supramolecular strategy modulating the property of the lipidic lamellar phase constitutes a new approach for increasing the delivery of various types of nucleic acid molecules. [ABSTRACT FROM AUTHOR]

This paper describes the formulation of archaeosomes and the evaluation of their abilities to facilitatein vitroDNA delivery. Lipids of theH.hispanica2TK2 strain were used in archaeosome formation, which is formulated by mixingH.hispanica2TK2 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 reagentsin vitro. [ABSTRACT FROM AUTHOR]

The association of liposome-based gene delivery systems with different biopolymers has been investigated to provide multifunctional properties for its complexes, such as alternative delivery routes and targeting delivery. Hyaluronic acid (HA) is an anionic mucoadhesive biopolymer that can interact to cell surface receptors. In this study, we explored the colloidal formation of pseudo-ternary complexes based on the electrostatic complexation among plasmid DNA (pDNA), cationic liposomes (CL) and hyaluronic acid of low molecular weight (16 kDa). We used extruded cationic liposomes composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (molar proportion of 2:1:1, respectively), pDNA vector model with luciferase as reporter gene, and HA of low molecular weight of 16 kDa. The pseudo-ternary complexes were physicochemically characterized in terms of their hydrodynamic diameter, polydispersity, zeta potential, morphology and accessibility to pDNA probe. These results together indicated that the pDNA/CL complexes are most likely coated by HA molecules with the pDNA incorporated into the liposomal structure. To evaluate the biological viability of the complexes, we performed in vitro transfections of human epitheloid carcinoma (HeLa) cells. The pDNA/CL/HA complexes presented transfection efficiencies similar to the pseudo-binary pDNA/CL complexes for the different HA amounts studied. In addition, we carried out time-lapse imaging in living HeLa cells so we could monitor the kinetics of complexes internalization. We believe that these results may be useful for future developments of multifunctional nanocarriers that explore the association of materials with different net charge properties for gene delivery and vaccine therapy applications. [ABSTRACT FROM AUTHOR]

Polyethylenimine (pEI) is a potent cationic transfection agent that is commonly used in conjunction with other inorganic and organometallics formulations. However, the toxicity that is intrinsic to pEI constitutes a major obstacle for both in vitro and in vivo applications. In this study, the roles of pEI molecular architectures and effects of nitrogen/phosphate (N/P) molar ratio on the cytotoxic potential and in vitro transfection performance were elucidated utilizing linear (L-pEI) and branched (B-pEI) 25 kDa pEI. Both L-pEI and B-pEI were able to self-assemble with the plasmid DNA to form positively charged nanoplexes having an average hydrodynamic diameter of 100-150 nm. In vitro cytotoxicity and transfection performance were assessed with three model mammalian cell lines namely, L929, COS-7 and HeLa cells. Transfection profiles over a wide range of N/P ratio exemplify a similar trend across three different cell lines, with B-pEI nanoplexes consistently showing maximum transgene expression of enhanced green fluorescent protein at an earlier N/P ratio compared to L-pEI nanoplexes. However, high transfection activity was maintained over a wider N/P ratio for L-pEI nanoplexes. While the absolute size of the nanoplexes exhibited no discernible effect on the transfection performance, cytotoxicity of free un-bound B-pEI was identified as the dictating factor responsible for the observed global cellular response. Collectively, our findings provided critical insights into the role of pEI molecular architecture in cellular transfection and are expected to lay the foundation for the rational design and development of advanced pEI-based inorganic and organometallic gene delivery systems. [ABSTRACT FROM AUTHOR]

We report the development of an improved in vitro transfection assay to test the efficiency of non-viral vector DNA nanoparticle transfection of primary hepatocytes. The protocol describes the isolation of viable hepatocytes from a mouse by collagenous perfusion. Primary mouse hepatocytes are plated in 384-well plates and cultured for 24 h prior to transfection with polyethylenimine (PEI) or peptide DNA nanoparticles. Luciferase expression is measured after 24 h following the addition of ONE-Glo substrate. The gene transfer assay for primary hepatocytes was optimized for cell plating number, DNA dose, and PEI to DNA ratio. The assay was applied to compare the expression mediated by mRNA relative to two plasmids possessing different promoters. The reported assay provides reliable in vitro expression results that allow direct comparison of the efficiency of different non-viral gene delivery vectors. [Display omitted] • Miniaturized efficient gene transfer protocol in primary hepatocytes is described providing large capacity. • Detailed parameters to achieve PEI mediated gene transfer of primary hepatocytes were developed. • Comparison of PEI and peptide mediated gene transfer of primary hepatocytes reveals significant differences. [ABSTRACT FROM AUTHOR]

Abstract: Plasmid DNA (pDNA) was efficiently encapsulated into biocompatible microgels by an inverse microemulsion polymerization method using ethylene glycol diglycidyl ether (EGDE) as cross-linker. Plasmid DNA and an anticancer drug, doxorubicin (DOX), could both be loaded with high encapsulation efficiency and released from pDNA microgels; quite relevant is also the less toxic effect of the incorporated drug, as compared with naked DOX. Photodisruption of microgels can be used as a strategy to enhance release. Mathematical models were applied to fully characterize the pDNA and drug release profiles, after light irradiation and at dark conditions; significant differences were find between the two conditions. As demonstrated by gel electrophoresis, the released plasmid DNA was intact. Moreover, the delivery of the encapsulated pDNA shows the capability of cell internalization and transfection in vitro resulting in the expression of the p53 protein. Additionally, a fluorymetry analysis demonstrated sustained pDNA release with microgels. The effect of this system on cell viability inhibition was evaluated in cancer Hela cells. The treatment with a pDNA/doxorubicin loaded microgel improves cell apoptosis. Compared with pDNA microgel or free drug, the co-delivery system has a stronger potential to suppress the development of cancer cells. We then present a quite promising pDNA based carrier that is biocompatible, photodegradable and delivers pDNA and probably different drugs, in a controlled and sustained manner; we hope this system can found interesting clinical uses, especially in the cancer treatment through a combined action of chemotherapy and gene delivery. [Copyright &y& Elsevier]

Highlights: [•] Self-assembled NMCTS-graft-PAMAM/pDNA complex was prepared. [•] The copolymer effectively formed spherical nanoparticles with pDNA. [•] DNA protection capability of chitosan was much improved after copolymerization. [•] The copolymer showed low toxicity and excellent blood compatibility. [•] Transfection efficiency of copolymer was significantly increased than PEI in HeLa cell. [ABSTRACT FROM AUTHOR]

Primary smooth muscle cells (SMC) isolated from the aorta of fetal calf were transfected with a green fluorescent protein (GFP)-encoding plasmid DNA, which was carried by a water-soluble and temperature-sensitive N-isopropylacrylamide-based (NIPAAm-based)-co-polymer, either poly(N-isopropylacrylamide-co-2-methacryloamidohistidine) (poly(NIPAAm-co-MAH)) or monosized PEGylated nanoparticle poly(styrene/poly(ethylene glycol) ethyl ether methacrylate/N-(3-(dimethylamino)propyl) methacrylamide) (poly(St/PEG-EEM/DMAPM)). Poly(NIPAAm-co-MAH) co-polymer was synthesized by solution polymerization of n-isopropylacrylamide (NIPAAm) and 2-methacrylamidohistidine (MAH). Monosized cationic nanoparticles were produced by emulsifier-free emulsion polymerization of styrene, PEG ethyl ether methacrylate and N-[3-(dimethyl-amino) propyl] methacrylamide, in the presence of a cationic initiator, 2,2-azobis (2-methylpropionamidine) dihydrochloride. The structure of poly(St/PEG-EEM/DMAPM) and poly(NIPAAm-co-MAH) was confirmed by1 H-NMR and FT-IR spectroscopy. Particle size/size distribution and surface charges of both carriers were measured by Zeta Sizer. The LCST behavior of poly(NIPAAm-co-MAH) co-polymer was followed spectrophotometrically. Poly(St/PEG-EEM/DMAPM) nanoparticles, with an average size of 78 nm and zeta potential of 54.4 mV, and an average size of 200 nm with a zeta potential of 54.2 mV, and poly(NIPAAm-co-MAH) were used in the transfection studies. The cytotoxicity of the vectors was tested using the MTT method. According to conditions for the transfection study (polymer/cell ratio and polymer-cell incubation period), cell loss was only 4 and 15% with poly(St/PEG-EEM/DMAPM) sized 78 and 200 nm, respectively. Poly(NIPAAm-co-MAH) cytotoxicity was insignificant. Poly(NIPAAm-co-MAH) uptake efficiency in SMCs was around 85%, but gene expression efficiency were low compared to poly(St/PEG-EEM/DMAPM)/pEGFP-N2 conjugates because of the low zeta potential of the co-polymer. Polymer uptake efficiencies of the nanoparticles were 90-95%. GFP expression efficiency was 68 and 64% after transfection with pEGFP-N2 conjugate with 78 and 200 nm sized poly(St/PEG-EEM/DMAPM) nanoparticles. [ABSTRACT FROM AUTHOR]

Abstract: Gemini surfactants are versatile gene delivery agents because of their ability to bind and compact DNA and their low cellular toxicity. Through modification of the alkyl tail length and the chemical nature of the spacer, new compounds can be generated with the potential to improve the efficiency of gene delivery. Amino acid (glycine and lysine) and dipeptide (glycyl-lysine and lysyl-lysine) substituted spacers of gemini surfactants were synthesized, and their efficiency of gene delivery was assessed in epithelial cells for topical cutaneous and mucosal applications. Three different epithelial cell lines, COS-7, PAM212 and Sf 1Ep cells, were transfected with plasmid DNA encoding for interferon gamma and green fluorescent protein complexed with the amino acid-substituted gemini compounds in the presence of 1,2 dioleyl-sn-glycero-phosphatidyl-ethanolamine as a helper lipid. Gene expression was quantified by ELISA. Size, zeta potential and circular dichroism measurements were used to characterize the plasmid–gemini (PG) and plasmid–gemini surfactant–helper lipid (PGL) complexes. Gene expression was found to increase up to 72h and then declined by the 7th day. In general, the glycine-substituted surfactant showed consistently high gene expression in all three cell lines. Results of physicochemical and spectroscopic studies of the complexes indicate that substitution of the gemini spacer does not interfere with compaction of the DNA. The superior performance of these spacer-substituted gemini surfactants might be attributed to their better biocompatibility compared to the surfactants possessing unsubstituted spacers. [Copyright &y& Elsevier]

Abstract: RDS (retinal degeneration slow) is a photoreceptor-specific tetraspanin protein required for the biogenesis and maintenance of rod and cone outer segments. Mutations in the Rds gene are associated with multiple forms of rod- and cone-dominant retinal degeneration. To gain more insight into the mechanisms underlying the regulation of this gene, the identification of regulatory sequences within the promoter of Rds was undertaken. A 3.5 kb fragment of the 5′ flanking region of the mouse Rds gene was isolated and binding sites for Crx, Otx2, Nr2e3, RXR family members, Mef2C, Esrrb, NF1, AP1, and SP1 in addition to several E-boxes, GC-boxes and GAGA-boxes were identified. Crx binding sequences were conserved in all mammalian species examined. Truncation expression analysis of the Rds promoter region in Y-79 retinoblastoma cells showed maximal activity in the 350 bp proximal promoter region. We also show that inclusion of more distal fragments reduced promoter activity to the basal level, and that the promoter activities are cell-type and direction specific. Co-transfection with Nrl increased promoter activity, suggesting that this gene positively regulates Rds expression. Based on these findings, a relatively small fragment of the Rds promoter may be useful in future gene transfer studies to drive gene expression in photoreceptors. [Copyright &y& Elsevier]

Abstract: Naked plasmid DNA is a powerful tool for gene therapy, but it is rapidly eliminated from the circulation after intravenous administration. Therefore, the development of optimized DNA delivery systems is necessary for its successful clinical use. Solid lipid nanoparticles (SLNs) have demonstrated transfection capacity in vitro, but their application for gene delivery has not been conveniently investigated in vivo. We aimed to evaluate the capacity of SLN–DNA vectors to transfect in vivo after intravenous administration to mice. The SLNs, composed of Precirol® ATO 5, DOTAP and Tween 80 were complexed with the plasmid pCMS-EGFP which encodes the enhanced green fluorescent protein (EGFP). The resulting systems were characterized in vitro showing a mean particle size of 276nm, superficial charge of +28mV, the ability to protect the plasmid and transfection capacity in culture cells. The intravenous administration in mice led to transfection in hepatic tissue and spleen. Protein expression was detected from the third day after administration, and it was maintained for at least 1 week. This work shows for the first time the capacity of SLN–DNA vectors to induce the expression of a foreign protein after intravenous administration, supporting the potential of SLNs for gene therapy. [Copyright &y& Elsevier]

Abstract: One of the major drawbacks of non-viral vector based gene delivery is the toxicity effects related to their positive charge. The aim of this work is to develop a cationic pullulan that can be used for gene delivery applications targeted to liver cells. Cationic groups were introduced by reacting varying amounts of glycidyl trimethyl ammonium chloride with pullulan. The cationic derivatives readily formed polyionic complexes with DNA and the size and zeta potential of these complexes were evaluated. The cytotoxicity and blood compatibility of cationic pullulan (CP) derivatives were assessed and compared to polyethyleneimine. Based on the cytotoxicity and blood compatibility test results CP3 was taken for further studies. Liver binding affinity of the modified pullulan CP3 was tested in vitro on hepatocytes and in vivo on mice. The complex was found to be stable in the presence of plasma as observed from gel retardation assay. This study focuses on the blood compatibility of the cationic pullulan, physico-chemical characterization and uptake of the nanocomplex by hepatocytes and in vitro transfection. [Copyright &y& Elsevier]

Abstract: Background: Recently, particle bombardment has become increasingly popular as a transfection method, because of a reduced dependency on target cell characteristics. In this study, we evaluated in vitro gene transfer by particle bombardment. Methods: gWIZ luciferase and gWIZ green fluorescent protein (GFP) plasmids were used as reporter genes. Mammalian cell lines HEK 293, MCF7 and NIH/3T3 were used in the transfection experiments. Transfection was performed by bombardment of the cells with gene-coated gold particles using the Helios Gene Gun. The technology was assessed by analyzing gene expression and cell damage. Cell damage was evaluated by MTT assay. Results: This technology resulted in efficient in vitro transfection, even in the cells which are difficult to transfect. The gene expression was dependent on the gene gun''s helium pressure, the sizes of the gold particles, the amount of the particles and DNA loading, while cell viability was mostly dependent on helium pressure and amount of the gold particles. Conclusions: This technology was useful to transfection of cells. Optimal transfection conditions were determined to be between 75 and 100 psi of helium pressure, 1.0 to 1.6 μm gold particle size and 0.5 mg of gold particle amount with a loading ratio of 4 μg DNA/mg gold particles. General significance: These findings will be useful in the design of gene gun device, and bring further improvements to the in vitro and in vivo transfection studies including gene therapy and vaccination. [Copyright &y& Elsevier]

Abstract: Fibrin based hydrogels have been employed in vitro as a scaffold to promote tissue formation and investigate underlying molecular mechanisms. These hydrogels support a variety of cellular processes, and are being developed to enhance the presentation of biological cues, or to tailor the biological cues for specific tissues. The presentation of these cues could alternatively be enhanced through gene delivery, which can be employed to induce the expression of tissue inductive factors in the local environment. This report investigates gene delivery within fibrin hydrogels for two in vitro models of tissue growth: i) cell encapsulation within and ii) cell seeding onto the hydrogel. Naked plasmid and lipoplexes can be efficiently entrapped within the hydrogel, and after 1 day in solution more than 70% of the entrapped DNA is retained within the gel, with a sustained release observed for at least 19 days. Encapsulated lipoplexes did not aggregate and retain their original size. Transgene expression in vitro by delivery of lipoplexes was a function of the fibrinogen and DNA concentration. For encapsulated cells, all cells had intracellular plasmid and transgene expression persisted for at least 10 days, with maximal levels achieved at day 1. For cell infiltration, expression levels were less than those observed for encapsulation, and expression increased throughout the culture period. The increasing expression levels suggest that lipoplexes retain their activity after encapsulation; however, interactions between fibrin and the lipoplexes likely limit internalization. The inclusion of non-viral vectors into fibrin-based hydrogels can be employed to induce transgene expression of encapsulated and infiltrating cells, and may be employed with in vitro models of tissue growth to augment the intrinsic bioactivity of fibrin. [Copyright &y& Elsevier]

Primary cell cultures from crustacea have been initiated since the 1960s, yet no permanent cell line is available. Primary cells have a limited proliferative capacity in culture due to cellular senescence, which is regulated by a group of dominant senescence genes. The aim of this research was to manipulate cell cycle regulation by transfecting Cherax quadricarinatus primary cells with oncogenes, in an effort to induce a permanent cell line. Human papillomaviruses (HPV) play a critical role in the formation of anogenital cancer. Research has demonstrated that the HPV-expressed E6 and E7 proteins function concomitantly to disrupt the p53 and retinoblastoma (Rb) tumor suppressor genes, regulators of the cell-cycle checkpoints at the first gap (G1) phase. HPV E6 and E7 genes were transfected into the C. quadricarinatus cells by lipofection. Successful transfection was demonstrated by the presence of oncogene messenger RNA by reverse transciptase polymerase chain reaction. At day 150, transfected cells still remain viable, although cell proliferation was stagnant. It may be that while transfection of the oncogenes was successful, no proliferation of the C. quadricarinatus cells was evident due to a lack of telomere maintenance. [ABSTRACT FROM AUTHOR]

Abstract: Chitosan nanoparticles have shown considerable promise as gene vectors but do not mediate transfection with satisfactory efficiency. To improve upon the transfection efficiency of chitosan, we approached the development of alginate–chitosan nanoparticles with the goals of maintaining low toxicity and biocompatibility. Through ionic gelation, particles were formed with a mean Z-average diameter of 157 nm and a zeta potential of +32 mV. Competition binding assays indicated that the presence of alginate reduces the strength of interaction between chitosan and DNA, contributing to improved transfection. Cell viability assays indicated that nanoparticles exhibit the same low toxicity as chitosan, and significantly reduced toxicity compared to a commercial liposome formulation. As well, complexation with nanoparticles maintained DNA integrity and protected it from nuclease degradation better than chitosan alone. Alginate–chitosan nanoparticles were able to mediate transfection of 293T cells four times that achieved by chitosan nanoparticles; at 48 h, the transfection efficiency was as high as with Lipofectamine™, with significantly reduced cytotoxicity. Overall, alginate inclusion improved the vector properties of chitosan-based nanoparticles, demonstrating superior transfection ability while maintaining biocompatibility and low toxicity. [Copyright &y& Elsevier]

Abstract: The objective of this study is to enhance the expression of a plasmid DNA for mesenchymal stem cells (MSC) by combination of 3-dimensional (3D) tissue engineered scaffolds and non-viral gene carrier. As a carrier of plasmid DNA, dextran-spermine cationic polysaccharide was prepared by means of reductive-amination between oxidized dextran and the natural oligoamine, spermine. As the MSC scaffold, collagen sponges reinforced by incorporation of poly(glycolic acid) (PGA) fibers were used. A complex of the cationized dextran and plasmid DNA of BMP-2 was impregnated into the scaffolds. MCS were seeded into each scaffold and cultured by a 3D culture method. When MSC were cultured in the PGA-reinforced sponge, the level of BMP-2 expression was significantly enhanced by the cationized dextran-plasmid DNA complex impregnated into the scaffold than by the cationized dextran-plasmid DNA complex in 2-dimensional (2D) (tissue culture plate) culture method. The alkaline phosphatase activity and osteocalcin content of transfected MSC cultured in the PGA-reinforced sponge were significantly higher compared with 2D culture method. We conclude that combination of cationized dextran plasmid DNA complex and 3D tissue engineered scaffold was promising to promote the in vitro gene expression for MSC. [Copyright &y& Elsevier]

Interleukin-2 (IL-2) expression plasmid (pCXWN-hIL-2) loaded chitosan microspheres were evaluated for using in gene-based immunotherapy. Chitosan microspheres containing pCXWN-hIL-2 were prepared by using a precipitation technique. In addition, the effects of different factors such as the concentration (0.35–0.70%) and the molecular weight of chitosan (low and medium molecular weights), the plasmid amount (5–10 μg/ml) and the presence of glutaraldehyde during the encapsulation process, on microsphere characteristics were investigated. The size of microspheres changed between 1.45 and 2.00 μm. All the formulation factors affected the size of microspheres. The structure of plasmid remained unchanged during the encapsulation process and the release studies. Plasmid encapsulation efficiency of chitosan microspheres was high (82–92%). The zeta potential values of microspheres was approximately +5.2 to +12.4 mV. In vitro release properties of microspheres changed with formulation variables. In vitro release of DNA changed with the concentration and molecular weight of chitosan and initial plasmid amount. Addition of glutaraldehyde is not necessary for a formulation. MAT-LyLu, the rat prostate adenocarcinoma cell line, was used for the determination of the in vitro transfectional activity of IL-2 encoding plasmid DNA loaded chitosan microspheres and the level of IL-2 expressed into the cells was assayed using a ELISA kit. High level of IL-2 expression was obtained with plasmid-loaded chitosan microspheres. Microspheres showed similar IL-2 production as lipofectin. The molecular weight of chitosan used and the amount of plasmid influenced the in vitro IL-2 production in the cells. Encapsulation of IL-2 encoding gene into chitosan microspheres might be a useful strategy to increase the expression and to control the delivery of cytokine gene to cells. [Copyright &y& Elsevier]

A new cationic emulsion system with high density was prepared increasing in vitro transfection efficiencies of adherent cells. Lipiodol with a density of 1.3 (g/ml) was selected to increase the density of the DNA/emulsion complex. Cationic lipid emulsions were formulated with mixtures of lipiodol and squalene as the oil phase and 1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane (DOTAP) as a cationic lipid. These emulsions were used to find the correlation between the density and the in vitro transfection efficiency. The physical characteristics of the new emulsion formulations were also determined. Heavier DNA/cationic lipid emulsion complex showed higher in vitro transfection efficiency on adherent cell lines in the presence of 10% serum compared to lighter ones. The cationic lipid emulsion formulated with lipiodol and DOTAP was more stable and showed better in vitro transfection efficiency than other carriers without lipiodol. Due to the high density of the carrier, the DNA/carrier complex sank to the bottom of the wells, thereby increasing the contact between the complex and adherent cells. The new lipiodol emulsion with high density showed superior transfection activities on adherent cells in the presence of serum. [Copyright &y& Elsevier]

In this study, we investigated different types of polyethyleneimine (PEI) and their block copolymers with N-isopropylacrylamide (NIPA) as temperature-sensitive polycationic non-viral vectors for transfection of HeLa cells in cell culture media. First carboxyl-terminated poly(NIPA) was synthesized and then copolymerized with PEIs branched or linear and with two different molecular weights (2 and 25 kDa). Addition of PEI units to the poly(NIPA) chains increased the LCST values up to body temperature. Zeta potentials of the copolymers were significantly lower than the corresponding PEI homopolymers. A green fluorescent protein expressing plasmid was used as a model. Complexes of this plasmid both with PEIs and their copolymers were formed. The zeta potentials of these complexes were between −3.1 and +21.3. Higher values were observed for the complexes prepared with branched and higher molecular weight PEIs. Copolymerization caused a profound decrease in the positive charges. Particle sizes of the complexes were in the range of 190–1235 nm. Using high polymer/plasmid ratios caused aggregation. The smallest complexes were obtained with the copolymer prepared with branched PEI with 25-kDa molecular weight. Copolymers were able to squeeze plasmid DNA more at the body temperature. Cytotoxicity was observed with PEIs especially with the branched higher molecular weights. Copolymerization reduced the cytotoxicity. The best in vitro DNA uptake efficiency (70%) was achieved with the complex prepared with poly(NIPA)/PEI25B. However, poly(NIPA)/PEI25L was the most successful vector for an effective gene expression without any significant toxicity. [Copyright &y& Elsevier]

The objective of this study is to investigate the efficiency of a non-viral gene carrier with RGD sequences, Pronectin F+ for gene transfection. The Pronectin F+ was cationized by introducing ethylenediamine (Ed), spermidine (Sd), and spermine (Sm) to the hydroxyl groups while the corresponding gelatin derivative was prepared similarly because gelatin also has one RGD sequence per molecule. The ζ potential and molecular size of Pronectin F+ and gelatin derivatives were examined before and after polyion complexation with a plasmid DNA of luciferase. When complexed with the plasmid DNA at the Pronectin F+/plasmid DNA mixing ratio of 50, the complex exhibited a ζ potential of about 10 mV, which is similar to that of the gelatin derivative–plasmid DNA complex. Irrespective of the type of Pronectin F+ and gelatin derivatives, their complexation enabled the apparent molecular size of plasmid DNA to reduce to about 200 nm, the size decreasing with the increased derivative/plasmid DNA weight mixing ratio. The rat gastric mucosal (RGM)-1 cells treated with both complexes exhibited significantly stronger luciferase activities than free plasmid DNA although the enhanced extent was significant for the Sm derivative compared with the corresponding Ed and Sd derivatives. Cell attachment was enhanced by the Pronectin F+ derivative to a significant high extent compared with the gelatin derivative. The amount of plasmid DNA internalized into the cells was enhanced by the complexation with every Pronectin F+ derivative compared with the gelatin derivative. For both of Pronectin F+ and gelatin carriers, the buffering capacity of Sm derivatives was higher than that of Ed and Sd derivatives and comparable to that of polyethyleneimine. It is likely that the high efficiency of gene transfection for the Sm derivative is due to the superior buffering effect. We conclude that the Sm derivative of Pronectin F+ is promising as a non-viral vector of gene transfection. [Copyright &y& Elsevier]

Topological isomers of plasmid DNA might be a factor affecting transfection efficiency. This study investigated the relationship between three major topoisomers of plasmid DNA, (supercoiled, open circular, and linear forms) and their biological activity. The three topoisomers of pDNA were transfected into CHO cells (Chinese Hamster Ovary) or SKnSH (human neuroblastoma) cells by (1) cationic liposomes, DOTAP/DOPE (1,2-dioleoyloxy-3-trimethylammonium propane/dioleolylphosphatidylethanolamine) or (2) electroporation. The open circular form of pCMV-luciferase demonstrated higher transfection efficiency (TE) than either supercoiled or linear while plasmid pGL3 showed no significant difference in TE for either delivery method. CHO and SKnSH cells showed similar patterns in transfection efficiency although the extent of expression was different. In order to test its stability in cell cytoplasmic lysate, pDNA was incubated at 37°C in NP40 lysate preparation. Surprisingly, supercoiled pDNA was the least stable in lysate solution (detected up to 4 min) whereas the open circular pDNA and linear form were degraded with a half-life value of 6 and 10 min respectively. To measure cellular associated pDNA, fluorescein labeled pDNA was complexed with DOTAP/DOPE liposome and transfected into CHO cells. The amount of pDNA associated with the cell was determined by flow cytometry. The flow cytometry studies demonstrated that the open circular form associated with the cell more than the linear forms when delivered with cationic liposomes. In conclusion, the three studied topoisomers of pDNA are transcriptionally active. The pattern of transgene production of the topoisoform of pDNA was independent of cell line and delivery method even though the level of expression was different. [ABSTRACT FROM AUTHOR]

Sindbis virus (SINV), a positive-sense single stranded RNA virus that causes mild symptoms in humans, is transmitted by mosquito bites. SINV reverse genetics have many implications, not only in understanding alphavirus transmission, replication cycle, and virus-host interactions, but also in biotechnology and biomedical applications. The rescue of SINV infectious particles is usually achieved by transfecting susceptible cells (BHK-21) with SINV-infectious mRNA genomes generated from cDNA constructed via in vitro translation (IVT). That procedure is time consuming, costly, and relies heavily on reagent quality. Here, we constructed a novel infectious SINV cDNA construct that expresses its genomic RNA in yeast cells controlled by galactose induction. Using spheroplasts made from this yeast, we established a robust polyethylene glycol-mediated yeast: BHK-21 fusion protocol to rescue infectious SINV particles. Our approach is timesaving and utilizes common lab reagents for SINV rescue. It could be a useful tool for the rescue of large single strand RNA viruses, such as SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Transfection by means of non-viral gene delivery vectors is the cornerstone of modern gene delivery. Despite the resources poured into the development of ever more effective transfectants, improvement is still slow and limited. Of note, the performance of any gene delivery vector in vitro is strictly dependent on several experimental conditions specific to each laboratory. The lack of standard tests has thus largely contributed to the flood of inconsistent data underpinning the reproducibility crisis. A way researchers seek to address this issue is by gauging the effectiveness of newly synthesized gene delivery vectors with respect to benchmarks of seemingly well-known behavior. However, the performance of such reference molecules is also affected by the testing conditions. This survey points to non-standardized transfection settings and limited information on variables deemed relevant in this context as the major cause of such misalignments. This review provides a catalog of conditions optimized for the gold standard and internal reference, 25 kDa polyethyleneimine, that can be profitably replicated across studies for the sake of comparison. Overall, we wish to pave the way for the implementation of standardized protocols in order to make the evaluation of the effectiveness of transfectants as unbiased as possible. [ABSTRACT FROM AUTHOR]

• RALA/pDNA vectors were developed at different N/P ratios. • Confocal microscopy studies confirmed pDNA uptake and nucleus localization. • In vitro transfection of HeLa cells leads to gene release and protein expression. • N/P ratio strongly tailors gene transfection efficiency and apoptosis. • The great asset of this vector relies on N/P ratio as a tailoring parameter. Cancer gene therapy based on p53 tumor suppressor gene supplementation emerges as one of the most challenging and promising strategies. The development of a suitable gene delivery system is imperative to ensure the feasibility and viability of cancer gene therapy in a clinical setting. The conception of delivery systems based on cell- penetrating peptides may deeply contribute for the evolution of therapy efficacy. In this context, the present work explores the p53 encoding plasmid DNA (pDNA) condensation ability of RALA peptide to produce a suitable intracellular delivery platform. These carriers, formed at several nitrogen to phosphate groups (N/P) ratio, were characterized in terms of morphology, size, surface charges, loading and complexation capacity and the fine structure has been analyzed by Fourier-transformed infrared (FTIR) spectroscopy. Confocal microscopy studies confirmed intracellular localization of nanoparticles, resulting in enhanced sustained pDNA uptake. Moreover, in vitro transfection of HeLa cells mediated by RALA/pDNA vectors allows for gene release and p53 protein expression. From these progresses, apoptosis in cancer cells has been investigated. It was found that N/P ratio strongly tailors gene transfection efficiency and, thus, it can be fine-tuned for desired degree of both protein expression and apoptosis. The great asset of the proposed system relies precisely on the use of N/P ratio as a tailoring parameter that can not only modulate vector´s properties but also the extent of pDNA delivery, protein expression and, consequently, the efficacy of p53 mediated cancer therapy. [ABSTRACT FROM AUTHOR]