Multi-armed poly(L-glutamic acid)-graft-oligoethylenimine copolymers as efficient nonviral gene delivery vectors

Background The application of polyethylenimine (PEI) in gene delivery has been severely limited by significant cytotoxicity that results from a nondegradable methylene backbone and high cationic charge density. It is therefore necessary to develop novel biodegradable PEI derivates for low-toxic, highly efficient gene delivery.Methods A series of novel cationic copolymers with various charge density were designed and synthesized by grafting different kinds of oligoethylenimine (OEI) onto a determinate multi-armed poly(L-glutamic acid) backbone. The molecular structures of multi-armed poly(L-glutamic acid)-graft-OEI (MP-g-OEI) copolymers were characterized using nuclear magnetic resonance, viscosimetry and gel permeation chromatography. Moreover, the MP-g-OEI/DNA complexes were measured by a gel retardation assay, dynamic light scattering and atomic force microscopy to determine DNA binding ability, particle size, zeta potential, complex formation and shape, respectively. MP-g-OEI copolymers were also evaluated in Chinese hamster ovary and human embryonic kidney-293 cells for their cytotoxicity and transfection efficiency.

A comparison of thiolated and disulfide‐crosslinked polyethylenimine for nonviral gene delivery

Branched polyethylenimine (25 kDa) is thiolated and compared with redox-sensitive crosslinked derivatives. Both polymers thiol contents are assessed; the thiolated polymers have 390–2300 mmol SH groups/mol, whereas the crosslinked polymers have lower thiol contents. Cytotoxicity assays show that both modified polymers give lower hemolysis than unmodified PEI. Increased thiol content increases gene transfer efficiency but also elevates cytotoxicity. Crosslinking improves plasmid DNA condensation and enhances transfection efficiency, but extensive crosslinking overstabilizes the polyplexes and decreases transfection, emphasizing the need to balance polyplex stabilization and unpacking. Thus, at low levels of crosslinking, 25 kDa PEI can be an efficient redox-sensitive carrier system.

Nonviral Gene Delivery of Growth and Differentiation Factor 5 to Human Mesenchymal Stem Cells Injected into a 3D Bovine Intervertebral Disc Organ Culture System

Intervertebral disc (IVD) cell therapy with unconditioned 2D expanded mesenchymal stem cells (MSC) is a promising concept yet challenging to realize. Differentiation of MSCs by nonviral gene delivery of growth and differentiation factor 5 (GDF5) by electroporation mediated gene transfer could be an excellent source for cell transplantation. Human MSCs were harvested from bone marrow aspirate and GDF5 gene transfer was achieved by in vitro electroporation. Transfected cells were cultured as monolayers and as 3D cultures in 1.2% alginate bead culture. MSC expressed GDF5 efficiently for up to 21 days. The combination of GDF5 gene transfer and 3D culture in alginate showed an upregulation of aggrecan and SOX9, two markers for chondrogenesis, and KRT19 as a marker for discogenesis compared to untransfected cells. The cells encapsulated in alginate produced more proteoglycans expressed in GAG/DNA ratio. Furthermore, GDF5 transfected MCS injected into an IVD papain degeneration organ culture model showed a partial recovery of the GAG/DNA ratio after 7 days. In this study we demonstrate the potential of GDF5 transfected MSC as a promising approach for clinical translation for disc regeneration.

Water-soluble fullerene (C60) derivatives as nonviral gene-delivery vectors.

A new class of water-soluble C60 transfecting agents has been prepared using Hirsch-Bingel chemistry and assessed for their ability to act as gene-delivery vectors in vitro. In an effort to elucidate the relationship between the hydrophobicity of the fullerene core, the hydrophilicity of the water-solubilizing groups, and the overall charge state of the C60 vectors in gene delivery and expression, several different C60 derivatives were synthesized to yield either positively charged, negatively charged, or neutral chemical functionalities under physiological conditions. These fullerene derivatives were then tested for their ability to transfect cells grown in culture with DNA carrying the green fluorescent protein (GFP) reporter gene. Statistically significant expression of GFP was observed for all forms of the C60 derivatives when used as DNA vectors and compared to the ability of naked DNA alone to transfect cells. However, efficient in vitro transfection was only achieved with the two positively charged C60 derivatives, namely, an octa-amino derivatized C60 and a dodeca-amino derivatized C60 vector. All C60 vectors showed an increase in toxicity in a dose-dependent manner. Increased levels of cellular toxicity were observed for positively charged C60 vectors relative to the negatively charged and neutral vectors. Structural analyses using dynamic light scattering and optical microscopy offered further insights into possible correlations between the various derivatized C60 compounds, the C60 vector/DNA complexes, their physical attributes (aggregation, charge) and their transfection efficiencies. Recently, similar Gd@C60-based compounds have demonstrated potential as advanced contrast agents for magnetic resonance imaging (MRI). Thus, the successful demonstration of intracellular DNA uptake, intracellular transport, and gene expression from DNA using C60 vectors suggests the possibility of developing analogous Gd@C60-based vectors to serve simultaneously as both therapeutic and diagnostic agents.

NonViral Gene Delivery of Growth and Differentiation Factor 6 (GDF6) to whole bovine Intervertebral Disc

Question: Low back pain is an increasing global health problem, which is associated with intervertebral disc (IVD) damage and de- generation. Major changes occur in the nucleus pulposus (NP), with the degradation of the extracellular matrix (ECM) [1]. Further studies showed that growth factors from the transforming growth factor (TGF) and bone morphogenic proteins (BMP) family may induce chondrogenic differentiation of mesenchymal stem cells (MSC) [2]. Focusing on non-viral gene therapies and their possible translation into the clinics, we investigated if GDF6 (syn. BMP13 or CDMP2) can induce regeneration of degraded NP. We hypothesized that IVD transfected with plasmid over-expressing GDF6 also up-regulates other NP- and chondrogenic cell markers and enhances ECM deposition. Methods: Bovine IVD cells were isolated by pronase/collagenase II overnight digestion. After monolayer expansion up to passage 3, cells were transfected with the plasmid pGDF6 (RG211366, Origene, SF) or with green fluorescence protein (GFP) control using the NeonÒ transfection system (Invitrogen, Basel), both equipped with a Cy- tomegalovirus (CMV) promotor to induce over-expression. We tested a range of yet unpublished parameters for each of the primary disc cells to optimize efficiency. To test a non-viral gene therapy applied directly to 3D whole organ culture, bovine IVDs were harvested from fresh tails obtained from the abattoir within 5 h post-mortem [3]. Discs were then pre-incubated for 24 h in high glucose Dulbecco’s Modified Eagle Medium and 5 % fetal calf serum. Each disc was transfected by injection of 5 lg of plasmid GDF6 (Origene, RG211366) into the center by 25G needle and using Hamilton sy- ringe. Electroporation was performed using 2-needle array electrode or tweezertrodes; 8 pulses at 200mv/cm with an interval of 10 ms were applied using ECM830 Square Wave Electroporation System (Harvard Apparatus, MA) (Fig. 1). After transfection discs were cultured for 72 h to allow expression of GFP or GDF6. Discs were then fixed, cryosectioned and analysed by immunofluorescence against GDF6. Results: We successfully transfected bovine NP and AF cells in monolayer culture with the two plasmids using a 1,400 V, 20 ms and 2 pulses with a *25 % efficiency using 0.15 M cells and 3 lg DNA (Fig. 1). Organ IVD culture transfection revealed GFP6 positive staining in the centre of the disc using 2-needle array electrode. Results from tweezertrodes did not show any GFP posi- tive cells. Conclusions: We identified novel parameters to successfully transfect primary bovine IVD cells. For transfection of whole IVD explants electroporation parameters need to be further optimized. Acknowledgments: This study was supported by the Lindenhof Foundation ‘‘Forschung und Lehre’’ (Project no. 13-02-F). References 1. Roughly PJ (2004) Spine (Phila) 29:2691–2699 2. 3. Clarke LE, McConell JC, Sherratt MJ, Derby B, Richardson SM, Hoyland JA (2014) Arthritis Res Ther 16:R67 Chan SC, Gantenbein-Ritter B (2012) J Vis Exp 60(60):e3490

Dendritic Poly(ether imine) Based Gene Delivery Vector

The nonviral vector based gene delivery approach is attractive due to advantages associated with molecular-level modifications suitable for optimization of vector properties. In a new class of nonviral gene delivery systems, we herein report the potential of poly(ether Mine) (PETIM) dendrimers to mediate an effective gene delivery function. PETIM dendrimer, constituted with tertiary amine branch points, n-propyl ether linkers and primary amines at their peripheries, exhibits significantly reduced toxicities, over a broad concentration range. The dendrimer complexes pDNA effectively, protects DNA from endosomal damages, and delivers to the cell nucleus. Gene transfection studies, utilizing a reporter plasmid pEGFP-C1 and upon complexation with dendrimer, showed a robust expression of the encoded protein. The study shows that PETIM dendrimers are hitherto unknown novel gene delivery vectors, combining features of poly(ethylene imine)-based polymers and dendrimers, yet are relatively nontoxic and structurally precise.

Targeting αvβ3 and α5β1 for gene delivery to proliferating VSMCs: synergistic effect of TGF-β1

TGF-beta1 levels increase after vascular injury and promote vascular smooth muscle cell (VSMC) proliferation. We define a nonviral gene delivery system that targets alphavbeta3 and alpha5beta1 integrins that are expressed on proliferating VSMCs and strongly induced by TGF-beta1. A 15-amino acid RGDNP-containing peptide from American Pit Viper venom was linked to a Lys(16) peptide as vector (molossin vector) and complexed with Lipofectamine or fusogenic peptide for delivery of luciferase or beta-galactosidase reporter genes to primary cultures of human, rabbit, and rat VSMCs. Preincubation of VSMCs with TGF-beta1 for 24 h, but not with PDGF-BB, interferon-gamma, TNF-alpha, nor PMA, increased alphavbeta3 and alpha5beta1 expressions on VSMCs and enhanced gene delivery of molossin vector. Thus beta-galactosidase activity increased from 35 +/- 5% (controls) to 75 +/- 5% after TGF-beta1 treatment, and luciferase activity increased fourfold over control values. Potential use of this system in vessel bypass surgery was examined in an ex vivo rat aortic organ culture model after endothelial damage. Molossin vector system delivered beta-galactosidase to VSMCs in the vessel wall that remained for up to 12 days posttransfection. The molossin vector system, when combined with TGF-beta1, enhances gene delivery to proliferating VSMCs and might have clinical applications for certain vasculoproliferative diseases.

Physical factors affecting plasmid DNA compaction in stearylamine-containing nanoemulsions intended for gene delivery

Cationic lipids have been used in the development of non-viral gene delivery systems as lipoplexes. Stearylamine, a cationic lipid that presents a primary amine group when in solution, is able to compact genetic material by electrostatic interactions. In dispersed systems such as nanoemulsions this lipid anchors on the oil/water interface confering a positive charge to them. The aim of this work was to evaluate factors that influence DNA compaction in cationic nanoemulsions containing stearylamine. The influence of the stearylamine incorporation phase (water or oil), time of complexation, and different incubation temperatures were studied. The complexation rate was assessed by electrophoresis migration on agarose gel 0.7%, and nanoemulsion and lipoplex characterization was done by Dynamic Light Scattering (DLS). The results demonstrate that the best DNA compaction process occurs after 120 min of complexation, at low temperature (4 ± 1 °C), and after incorporation of the cationic lipid into the aqueous phase. Although the zeta potential of lipoplexes was lower than the results found for basic nanoemulsions, the granulometry did not change. Moreover, it was demonstrated that lipoplexes are suitable vehicles for gene delivery. © 2012 by the authors; licensee MDPI, Basel, Switzerland.

In vivo electroporation mediated gene delivery to the beating heart

Gene therapy may represent a promising alternative strategy for cardiac muscle regeneration. In vivo electroporation, a physical method of gene transfer, has recently evolved as an efficient method for gene transfer. In the current study, we investigated the efficiency and safety of a protocol involving in vivo electroporation for gene transfer to the beating heart. Adult male rats were anesthetised and the heart exposed through a left thoracotomy. Naked plasmid DNA was injected retrograde into the transiently occluded coronary sinus before the electric pulses were applied. Animals were sacrificed at specific time points and gene expression was detected. Results were compared to the group of animals where no electric pulses were applied. No post-procedure arrhythmia was observed. Left ventricular function was temporarily altered only in the group were high pulses were applied; CK-MB (Creatine kinase) and TNT (Troponin T) were also altered only in this group. Histology showed no signs of toxicity. Gene expression was highest at day one. Our results provide evidence that in vivo electroporation with an optimized protocol is a safe and effective tool for nonviral gene delivery to the beating heart. This method may be promising for clinical settings especially for perioperative gene delivery.

Chitosan-modified dry powder formulations for pulmonary gene delivery

Spray-drying is an effective process for preparing micron-dimensioned particles for pulmonary delivery. Previously, we have demonstrated enhanced dispersibility and fine particle fraction of spray-dried nonviral gene delivery formulations using amino acids or absorption enhancers as dispersibility-enhancing excipients. In this study, we investigate the use of the cationic polymer chitosan as a readily available and biocompatible dispersibility enhancer. Lactose-lipid:polycation:pDNA (LPD) powders were prepared by spray-drying and post-mixed with chitosan or spray-dried chitosan. In addition, the water-soluble chitosan derivative, trimethyl chitosan, was added to the lactose-LPD formulation before spray-drying. Spray-dried chitosan particles, displaying an irregular surface morphology and diameter of less than 2 mu m, readily adsorbed to lactose-LPD particles following mixing. In contrast with the smooth spherical surface of lactose-LPD particles, spray-dried trimethyl chitosan-lactose-LPD particles demonstrated increased surface roughness and a unimodal particle size distribution (mean diameter 3.4 mu m), compared with the multimodal distribution for unmodified lactose-LPD powders (mean diameter 23.7 mu m). The emitted dose and in vitro deposition of chitosan-modified powders was significantly greater than that of unmodified powders. Moreover, the inclusion of chitosan mediated an enhanced level of reporter gene expression. In summary, chitosan enhances the dispersibility and in vitro pulmonary deposition performance of spray-dried powders.

In vivo gene delivery to the liver using reconstituted chylomicron remnants as a novel nonviral vector

Lipoproteins are emulsion particles that consist of lipids and apolipoproteins. Their natural function is to transport lipids and/or cholesterol to different tissues. We have taken advantage of the hydrophobic interior of these natural emulsions to solubilize DNA. Negatively charged DNA was first complexed with cationic lipids containing a quaternary amine head group. The resulting hydrophobic complex was extracted by chloroform and then incorporated into reconstituted chylomicron remnant particles (≈100 nm in diameter) with an efficiency ≈65%. When injected into the portal vein of mice, there were ≈5 ng of a transgene product (luciferase) produced per mg of liver protein per 100 μg injected DNA. This level of transgene expression was ≈100-fold higher than that of mice injected with naked DNA. However, such a high expression was not found after tail vein injection. Histochemical examination revealed that a large number of parenchymal cells and other types of cells in the liver expressed the transgene. Gene expression in the liver increased with increasing injected dose, and was nearly saturated with 50 μg DNA. At this dose, the expression was kept at high level in the liver for 2 days and then gradually reduced and almost disappeared by 7 days. However, by additional injection at day 7, gene expression in the liver was completely restored. By injection of plasmid DNA encoding human α1-antitrypsin, significant concentrations of hAAT were detected in the serum of injected animals. This is the first nonviral vector that resembles a natural lipoprotein carrier.

Dendron conjugation to graphene oxide using click chemistry for efficient gene delivery

Owing to its large surface area and rapid cellular uptake, graphene oxide (GO) is emerging as an attractive candidate material for delivery of drugs and genes. The inherent sp(2) pi-pi interaction of GO helps to carry drugs and single stranded RNA (ssRNA) but there is no such interaction with double stranded DNA (dsDNA). In this work, a polyamidoamine (PAMAM) dendron was conjugated with nano GO (nGO) through ``click'' chemistry to improve the DNA complexation capability of GO as well as its transfection efficiency. The DNA complexation capability of GO was significantly enhanced after dendronization of GO yielding spherical nanosized (250-350 nm) particles of the dendronized GO (DGO)/pDNA complex with a positive zeta potential. The transfection efficiency of GO dramatically increased after conjugation of the PAMAM dendron. Transfection efficiency of 51% in HeLa cells with cell viability of 80% was observed. The transfection efficiency was significantly higher than that of polyethyleneimine 25 kDa (27% efficiency) and also surpassed that of lipofectamine 2000 (47% efficiency). The uptake of the DGO/pDNA complex by the caveolae mediated endocytosis pathway may significantly contribute to the high transfection efficiency. Thus, dendronized GO is shown to be an efficient gene carrier with minimal toxicity and is a promising candidate for use as a nonviral carrier for gene therapy.

Biofunctionalized surface-modified silver nanoparticles for gene delivery

Silver nanoparticles (AgNPs) find use in different biomedical applications including wound healing and cancer. We propose that the efficacy of the nanoparticles can be further augmented by using these particles for gene delivery applications. The objective of this work was to engineer biofunctionalized stable AgNPs with good DNA binding ability for efficient transfection and minimal toxicity. Herein, we report on the one-pot facile green synthesis of polyethylene glycol (PEG) stabilized chitosan-g-polyacrylamide modified AgNPs. The size of the PEG stabilized AgNPs was 38 +/- 4 nm with a tighter size distribution compared to the unstabilized nanoparticles which showed bimodal distribution of particle sizes of 68 +/- 5 nm and 7 +/- 4 nm. To enhance the efficiency of gene transfection, the Arg-Gly-Asp-Ser (RGDS) peptide was immobilized on the silver nanoparticles. The transfection efficiency of AgNPs increased significantly after immobilization of the RGDS peptide reaching up to 42 +/- 4% and 30 +/- 3% in HeLa and A549 cells, respectively, and significantly higher than 34 +/- 3% and 23 +/- 2%, respectively, with the use of polyethyleneimine (25 kDa). These nanoparticles were found to induce minimal cellular toxicity. Differences in cellular uptake mechanisms with RGDS immobilization resulting in improved efficiency are elucidated. This study presents biofunctionalized AgNPs for potential use as efficient nonviral carriers for gene delivery with minimal cytotoxicity toward augmenting the therapeutic efficacy of AgNPs used in different biomedical products.

Synergistic effects on gene delivery - co-formulation of small disulfide-linked dendritic polycations with Lipofectamine 2000 (TM)

This paper describes the application of gene delivery vectors based on connecting together two well-defined low-generation poly(L-lysine) (PLL) dendrons using a disulfide-containing linker unit. We report that the transfection ability of these vectors in their own right is relatively low, because the low-generation number limits the endosomal buffering capacity. Importantly, however, we demonstrate that when applied in combination with Lipofectamine 2000 (TM), a vector from the cationic lipid family, these small cationic additives significantly enhance the levels of gene delivery (up to four-fold). Notably, the cationic additives have no effect on the levels of transfection observed with a cationic polymer, such as DEAE dextran. We therefore argue that the synergistic effects observed with Lipofectamine 2000 (TM) arise as a result of combining the delivery advantages of two different classes of vector within a single formulation, with our dendritic additives providing a degree of pH buffering within the endosome. As such, the data we present indicate that small dendritic structures, although previously largely overlooked for gene delivery owing to their inability to transfect in their own right, may actually be useful well-defined additives to well-established vector systems in order to enhance the gene delivery payload.