Increased Transfection Efficiency Research Articles

RNA Interference Screen to Identify Pathways That Enhance or Reduce Nonviral Gene Transfer During Lipofection

Some barriers to DNA lipofection are well characterized; however, there is as yet no method of finding unknown pathways that impact the process. A druggable genome small-interfering RNA (siRNA) screen against 5,520 genes was tested for its effect on lipofection of human aortic endothelial cells (HAECs). We found 130 gene targets which, when silenced by pooled siRNAs (three siRNAs per gene), resulted in enhanced luminescence after lipofection (86 gene targets showed reduced expression). In confirmation tests with single siRNAs, 18 of the 130 hits showed enhanced lipofection with two or more individual siRNAs in the absence of cytotoxicity. Of these confirmed gene targets, we identified five leading candidates, two of which are isoforms of the regulatory subunit of protein phosphatase 2A (PP2A). The best candidate siRNA targeted the PPP2R2C gene and produced a 65% increase in luminescence from lipofection, with a quantitative PCR-validated knockdown of approximately 76%. Flow cytometric analysis confirmed that the silencing of the PPP2R2C gene resulted in an improvement of 10% in transfection efficiency, thereby demonstrating an increase in the number of transfected cells. These results show that an RNA interference (RNAi) high-throughput screen (HTS) can be applied to nonviral gene transfer. We have also demonstrated that siRNAs can be co-delivered with lipofected DNA to increase the transfection efficiency in vitro.

An Acid Sensitive Ketal-Based Polyethylene Glycol-Oligoethylenimine Copolymer Mediates Improved Transfection Efficiency at Reduced Toxicity

Dynamic PEG-polycation copolymers that release PEG and degrade into small fragments after cell entry might present efficient and biocompatible gene carriers. PEG-OEI-MK was synthesized by copolymerization of 5 kDa polyethyleneglycol (PEG) and 800 Da oligoethylenimines through acid-degradable acetone-bis-(N-maleimidoethyl)ketal linkers (MK). To evaluate any benefit of the reversible over stable linkage, also the corresponding pH-stable analog, PEG-OEI-BM, was synthesized via ether linkages. Luciferase and GFP expression plasmids were used for transfections, in vivo biocompatibility was evaluated by intravenous application of polymers in Balb/c mice. PEG-OEI-MK showed efficient DNA binding as analyzed by ethidium bromide exclusion, resulting in formation of polyplexes with sizes around 100 nm and surface charges of below 5 mV zeta potential. This surface shielding of PEG-OEI-MK polyplexes remained stable at neutral pH 7.4, while polyplexes deshielded and aggregated at pH 5 within 15-30 min. Cell culture experiments demonstrated reduced polymer toxicity compared to the non-PEGylated OEI-MK. Transfection experiments demonstrated reduced gene expression of PEG-OEI-BM compared with the non-PEGylated analog OEI-BM, whereas the pH-reversible polymer PEG-OEI-MK mediated a significant increased transfection efficiency over the non-PEGylated OEI-MK. PEG-OEI-MK mediated the highest gene transfer at lowest cytotoxicity levels and also best in vivo biocompatibility.

Relating Chemical and Biological Diversity Space: A Tunable System for Efficient Gene Transfection

Polyethyleneimine (PEI), a well-established nonviral transfection reagent, was combinatorially modified with varying proportions of methyl, benzyl, and n-dodecyl groups to create a library of 435 derivatized polymers. Screening of this library for transfection, DNA binding, and toxicity allows systematic correlation of the biological properties of our polymers to their derivatizations. Combinations of derivatizations bring about a 100-fold variation in transfection efficiency between library members. The best PEI derivatives exhibit increases in transfection efficiency of more than 80-fold over unmodified PEI (up to 28+/-7 % of cells transfected) and rival commercial reagents such as Lipofectamine 2000 (21+/-10 %) and JetPEI (32+/-5.0 %). In addition, we can identify compounds that are specifically tuned for efficient transfection in CHO-K1 over Ishikawa cells and vice versa, demonstrating that the approach can lead to cell-type selectivity of at least one order of magnitude. This work demonstrates that multivalent derivatization of a polymeric framework can create functional diversity substantially greater than the structural diversity of the derivatization building blocks and suggests an approach to a better understanding of the molecular underpinnings of transfection as well as their exploitation.

Substance P-Mediated Expression of the Pro-Angiogenic Factor CCN1 Modulates the Course of Colitis

Substance P (SP) regulates important intestinal functions, such as mucosal permeability, motility, chloride secretion, and inflammation via the neurokinin-1 receptor (NK-1R). Previous reports showed that vascularization and expression of angiogenic factors are evident in the colonic mucosa of rats with colitis and patients with inflammatory bowel disease. Here we determined whether SP is associated with angiogenesis. Human NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells) and mice with dextran sodium sulfate-induced colitis were used. We found that expression of the angiogenic factor CCN1 was increased in the colons of patients with Crohn's disease and ulcerative colitis. Mucosal extracts from inflammatory bowel disease patients induced human intestinal microvascular endothelial cell migration that was inhibited by blockade of CCN1 and its receptor integrin alphavbeta3. Both the degree of angiogenesis and CCN1 expression were elevated in the colons of mice with dextran sodium sulfate-induced colitis, which was reduced by treatment with the NK-1R antagonist CJ-12255. SP also increased CCN1 expression in NCM460-NK-1R colonocytes. SP exposure to human intestinal microvascular endothelial cells co-cultured with NCM460-NK-1R cells induced angiogenic activity that was inhibited by CCN1 silencing. In addition, intracolonic overexpression of CCN1 induced angiogenesis in mouse colon. Thus, SP mediates angiogenesis via CCN1 during colitis.

Novel polymer carriers and gene constructs for treatment of myocardial ischemia and infarction

The number one cause of mortality in the US is cardiovascular related disease. Future predictions do not see a reduction in this rate especially with the continued rise in obesity [P. Poirier, et al., Obesity and cardiovascular disease: pathophysiology, evaluation, and effect of weight loss, Arterioscler Thromb Vasc Biol. 26(5), (2006) 968–976.; K. Obunai, S. Jani, G.D. Dangas, Cardiovascular morbidity and mortality of the metabolic syndrome, Med.Clin. North Am., 91(6), (2007) 1169–1184]. Even so, potential molecular therapeutic targets for cardiac gene delivery are in no short supply thanks to continuing advances in molecular cardiology. However, efficient and safe delivery remains a bottleneck in clinical gene therapy [O.J. Muller, H.A. Katus, R. Bekeredjian, Targeting the heart with gene therapy-optimized gene delivery methods, Cardiovasc Res, 73(3), (2007) 453–462]. Viral vectors are looked upon favorably for their high transduction efficiency, although their ability to elicit toxic immune responses remains [C.F. McTiernan, et al., Myocarditis following adeno-associated viral gene expression of human soluble TNF receptor (TNFRII-Fc) in baboon hearts, Gene Ther, 14(23), (2007) 1613–1622]. However, this high transduction does not necessarily translate into improved efficacy [X. Hao, et al., Myocardial angiogenesis after plasmid or adenoviral VEGF-A(165) gene transfer in rat myocardial infarction model, Cardiovasc Res., 73(3), (2007) 481–487]. Naked DNA remains the preferred method of DNA delivery to cardiac myocardium and has been explored extensively in clinical trials. The results from these trials have demonstrated efficacy in regard to secondary end-points of reduced symptomatology and perfusion, but have failed to establish significant angiogenesis or an increase in myocardial function [P.B. Shah, D.W. Losordo, Non-viral vectors for gene therapy: clinical trials in cardiovascular disease, Adv Genet, 54, (2005) 339–361]. This may be due in part to reduced transfection efficiency but can also be attributed to use of suboptimal candidate genes. Currently, polymeric non-viral gene delivery to cardiac myocardium remains underrepresented. In the past decade several advances in non-viral vector development has demonstrated increased transfection efficiency [O.J. Muller, H.A. Katus, R. Bekeredjian, Targeting the heart with gene therapy-optimized gene delivery methods, Cardiovasc Res, 73(3), (2007) 453–462]. Of these polymers, those that employ lipid modifications to improve transfection or target cardiovascular tissues have proven themselves to be extremely beneficial. Water-soluble lipopolymer (WSLP) consists of a low molecular weight branched PEI (1800) and cholesterol. The cholesterol moiety adds extra condensation by forming stable micellular complexes and was later employed for myocardial gene therapy to exploit the high expression of lipoprotein lipase found within cardiac tissue. Use of WSLP to deliver hypoxia-responsive driven expression of hVEGF to ischemic rabbit myocardium has proven to provide for even better expression in cardiovascular cells than Terplex and has demonstrated a significant reduction in infarct size (13 ± 4%, p < 0.001) over constitutive VEGF expression (32 ± 7%, p = 0.007) and sham-injected controls (48 ± 7%). A significant reduction in apoptotic values and an increase in capillary growth were also seen in surrounding tissue. Recently, investigations have begun using bioreducible polymers made of poly(amido polyethylenimines) (SS-PAEI). SS-PAEIs breakdown within the cytoplasm through inherent redox mechanisms and provide for high transfection efficiencies (upwards to 60% in cardiovascular cell types) with little to no demonstrable toxicity. In vivo transfections in normoxic and hypoxic rabbit myocardium have proven to exceed those results of WSLP transfections by 2–5 fold [L.V. Christensen, et al., Reducible poly(amido ethylenediamine) for hypoxia-inducible VEGF delivery, J Control Release, 118(2), (2007) 254–261]. This new breed of polymer(s) may allow for decreased doses and use of new molecular mechanisms not previously available due to low transfection efficiencies. Little development has been seen in the use of new gene agents for treatment of myocardial ischemia and infarction. Current treatment consists of using mitogenic factors, described decades earlier, alone or in combination to spur angiogenesis or modulating intracellular Ca 2+ homeostasis through SERCA2a but to date, failed to demonstrate clinical efficacy. Recent data suggests that axonal guidance cues also act on vasculature neo-genesis and provide a new means of investigation for treatment.

Folate-linked lipid-based nanoparticles for synthetic siRNA delivery in KB tumor xenografts

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism triggered by synthetic small interfering RNA (siRNA), and is utilized in a wide range of fields including cancer gene therapy by down-regulating a specific target protein. In this study, for tumor-targeted siRNA delivery, we developed a folate-linked nanoparticle (NP-F), and evaluated the potential of NP-F-mediated tumor gene therapy in human nasopharyngeal KB cells, which overexpressed folate receptor (FR). NP-F was composed of cholesteryl-3β-carboxyamidoethylene- N-hydroxyethylamine (OH-Chol), Tween 80 and folate-poly(ethylene glycol)-distearoylphosphatidylethanolamine conjugate (f-PEG 2000-DSPE), and NP-P was substituted f-PEG 2000-DSPE in NP-F PEG 2000-DSPE for a non-targeting nanoparticle. The NP-F and siRNA complex (nanoplex) formed at a charge ratio (+/−) of 2/1 in the presence of 5 mM NaCl was injectable size and increased transfection efficiency in the cells. NP-F showed a significantly higher intracellular amount of siRNA and stronger localization of siRNA in the cytoplasm than NP-P. When Her-2 siRNA was transfected into cells by NP-F and NP-P, NP-F significantly inhibited tumor growth, and selectively suppressed Her-2 protein expression more than NP-P. In in vivo gene therapy, a NP-F nanoplex of Her-2 siRNA by intratumoral injection significantly inhibited tumor growth of KB xenografts compared with control siRNA, but a NP-P nanoplex did not. These results of the experiments have provided optimal conditions to form folate-linked nanoparticle complexes with siRNA for folate-targeted gene therapy.

Preparation of Cationic Gold Nanoparticles for Gene Delivery

Cationic gold nanoparticles modified with 2-aminoethanethiol, 8-amino-1-octanethiol, and 11-amino-1- undecanethiol were prepared by NaBH4 reduction of HAuCl4 in the presence of thiols in water or a water/ethanol mixture solvent. The formation of gold nanoparticles depended on the kind of solvent, and the concentration of NaBH4, HAuCl4, and thiols. A low concentration of HAuCl4 and a high concentration of NaBH4 facilitated the formation of gold nanoparticles. A high concentration of HAuCl4 solution resulted in aggregation and a low concentration of NaBH4 did not induce the formation of gold nanoparticles. The size of the gold nanoparticles increased with an increase of the concentration of the HAuCl4 solution, while it decreased with an increase of the concentration of the NaBH4 solution. When thiols were present, the HAuCl4/thiol ratio must be in the range of 1:1 to 1:2 for the formation of gold nanoparticles in both the water and water/ethanol mixture solvents. Modification with thiol molecules changed surface charge of the gold nanoparticles from negative to positive, and stabilized the gold nanoparticles. The cationic gold nanoparticles promoted gene transfection. A high ratio of gold nanoparticles to DNA increased transfection efficiency. However, the length of the alkyl groups had no evident effect.

Analysis of Transgene Intergration Efficiencies into Porcine Fetal Fibroblast Using Different Transfection Methods and Twice Drug Selection.

Transgenic animals can be produced by nuclear transfer with genetically modified somatic cells in which the procedure of transgene transfection is required. The objective of this study was to compare transfection efficiencies of electroporation (Microporater [Seo Ryn Bio, Korea]) and liposome reagents (FuGENE 6, FuGENE HD [Roche, USA], Welfect-ex, Welfect-Q, Welfect-M [Welgene, Korea]) into pig fetal fibroblast cells using transgene vector containing human tPA cDNA fused to β-casein promoter sequence and neomycin-resistant gene (Neor) driven by PGK promoter and to determine the proportion of cells containing transgene in neomycin-resistant colonies. The linearized transgene vector DNA was transfected into pig fetal fibroblasts with electroporation or different transfection reagents according to manufacturer's procedures. Pig fetal fibroblast cells were selected following exposure to 400 μg/ml G418 for 14 days. Cell colonies surviving G418 selection were picked by cloning cylinder (Sigma, USA) and assayed by PCR amplification with tPA-specific primers. Also, single cell PCR analysis of growing colonies resistant to G418 selection was carried out to evaluate the percentage of cells containing transgene in G418-resistant colonies. Among transfection methods tested in this experiment, electroproation exhibited significantly higher transfection efficiency compared with liposome reagents (50% with microporater vs 20% with liposomes). When cells of G418-resistant colonies were performed by single-cell PCR, 60 to 100% of cells in each colony were identified to contain transgene regardless of transfection methods. The colonies carrying 60 to 80 % transgene-positive cells were cultured again with 400 μg/ml G418 for 14 days, and cell colonies surviving G418 selection were selected and assayed by PCR. Transfection frequencies in theses colonies for tPA gene were 95% to 100%. The present results suggested that electroproation may increase transfection efficiency of DNA into pig fetal fibroblast cells compared with liposome methods and twice drug-selection can improve transfection rate.

Silicalite nanoparticles that promote transgene expression

Here, we report on a new zeolite-based silicalite nanoparticle that can enhance thetransfection efficiencies generated by poly ethylene imine–plasmid DNA (PEI–pDNA)complexes via a sedimentation mechanism and can enhance the transfectionefficiencies of pDNA alone when surface functionalized with amine groups. Thesilicalite nanoparticles have a mean size of 55 nm. Functionalizing the silicalitenanoparticles with amine groups results in a clear transition in zeta potential from−25.9 ± 2.3 mV (pH 7.4) for unfunctionalized silicalite nanoparticles to4.9 ± 0.7 mV (pH 7.4) for amine functionalized silicalite nanoparticles. We identify thatsilicalite nanoparticles used to promote non-viral vector acceleration to the cellsurface are found in acidic vesicles or the cytoplasm but not the nucleus. An MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay showed that thesilicalite nanoparticles were non-toxic at the concentrations tested for transfection. Weshow that surface functionalization of silicalite nanoparticles with amine groupsresults in a significant (230%) increase in transfection efficiency of pDNA whencompared to unfunctionalized silicalite nanoparticles. Silicalite nanoparticles enhancedpDNA–PEI induced transfection of human embryonic kidney (HEK-293) cells by over150%.

Physicochemical Characterization of Thermoresponsive Poly(N-isopropylacrylamide)−poly(ethylene imine) Graft Copolymers

Synthetic polycations have shown promise as gene delivery vehicles but suffer from an unacceptable toxicity and low transfection efficiency. Novel architectures are being explored to increase transfection efficiency, including copolymers with a thermoresponsive character. The physicochemical characterization of a family of copolymers comprising a core of the cationic polymer poly(ethylene imine) (PEI) with differing thermoresponsive poly( N-isopropylacrylamide) (PNIPAM) grafts has been carried out using pulsed-gradient spin-echo NMR (PGSE-NMR) and small-angle neutron scattering (SANS). For the copolymers that have longer chain PNIPAM grafts, there is clear evidence of the collapse of the grafts with increasing temperature and the associated emergence of an attractive interpolymer interaction. These facets depend on the number of PNIPAM grafts attached to the PEI core. While a collapse in the smaller PNIPAM grafts is observed for the third polymer, there is no appearance of the interpolymer attractive interaction. These observations provide further insight into the association behavior of these copolymers, which is fundamental to developing a full understanding of how they interact with nucleic acids. Furthermore, the differing behaviors of the three copolymers over temperatures in which the PNIPAM blocks undergo coil-to-globule transitions is indicative of changes in the presentation of charged-core and hydrophobic chain components, which are key factors affecting nucleic acid binding and, ultimately, cell transfection ability.

Enhancing Polysaccharide-Mediated Delivery of Nucleic Acids Through Functionalization with Secondary and Tertiary Amines

Chitosan is a polysaccharide that has generated significant interest as a non-viral gene delivery vehicle due to its cationic and biocompatible characteristics. However, transfection efficiency of chitosan is significantly lower compared to other cationic gene delivery agents, e.g. polyethyleneimine (PEI), dendrimers or cationic lipids. This is primarily attributed to its minimal solubility and low buffering capacity at physiological pH leading to poor endosomal escape of the gene carrier and inefficient cytoplasmic decoupling of the complexed nucleic acid. Here we have developed an imidazole acetic acid (IAA)-modified chitosan to introduce secondary and tertiary amines to the polymer in order to improve its endosomal buffering and solubility. The modified polymer was characterized by ninhydrin and (1)H NMR assays for degree of modification, while buffering and solubility were analyzed by acid titration. Nanocomplex formation, studied at various polymer-nucleic acid ratios, showed an increase in particle zeta potential for chitosan-IAA, as well as an increase in the effective diameter. Up to 100-fold increase in transfection efficiency of pDNA was seen for chitosan-IAA as compared to native chitosan, nearly matching that of PEI. In addition, transfection of siRNA by the modified polymers showed efficient gene knockdown equivalent to commercially available siPORT Amines. Collectively, these results demonstrate the potential of the imidazole-grafted chitosan as a biocompatible and effective delivery vehicle for both pDNA and siRNA.

Ultrasonic imaging of molecular targets

Today nuclear medicine is the only modality that is clinically successful in molecular imaging. However, other modalities compete with its excellent sensitivity in imaging molecular targets. In the last 10 years ultrasound imaging has shown the potential to provide sufficiently high sensitivity for the molecular imaging of vascular targets. These advances are based on the joint development of microbubble contrast media and the methods to image them with high sensitivity. Ultrasound-contrast-enhanced imaging strategies make use of the specific physical properties of microbubbles such as resonance, nonlinear oscillation, and collapse. The size of microbubbles limits their use to the vascular space. Thus, the main applications of ultrasound for molecular imaging are inflammation, thrombi, and angiogenesis, for which successful contrast enhancement has been achieved in animal models. Main molecular targets used to date include selectins, alpha(v)beta(3) or alpha(5)beta(1) integrins, glycoprotein (GP) IIb/IIIa, intracellular adhesion molecule ICAM-1, and vascular endothelial growth factor receptor VEGFR2. Results from animal studies indicate that ultrasound could play a major role in vascular molecular imaging for diagnosis and treatment monitoring. Additional effects of insonified microbubbles (e.g., opening of the blood-brain barrier or increased transfection efficiency in gene therapy) are attributed to the transient opening of cell membranes known as "sonoporation" and demonstrate further potential for integrated diagnosis and therapy.

Optimization is Essential to Increase Transfection Efficiency and Cell Viability in Primary and Difficult‐to‐Transfect cell lines

Electroporation is a valuable tool for nucleic acid delivery because it can be used for a wide variety of cell types. Many scientists are shifting towards the use of cell types of increasing scientific interest, including lineage‐specific cell types, bone marrow cells and stem cells, but these cells can be difficult to transfect. The ability to transfect these cell types with nucleic acid molecules of interest at a relatively high efficiency while maintaining cell viability would offer a new tool towards the elucidation of the pathway(s) in which a gene product is involved.In this poster, we present data demonstrating that optimization of electroporation parameters is essential to deliver molecules in a highly efficient manner. We display results of transfection of several difficult to transfect cell types, including Human Primary Fibroblasts, HUVEC, Jurkat and (mouse) Neuro‐2A neural cells with plasmid DNAs and siRNAs. We used multiple detection methods to determine transfection efficiency, including flow cytometry, real‐time PCR and the luciferase assay. Gene Pulser electroporation buffer and square or exponential wave pulses were used to convey molecules of interest into the cells. Optimization of electroporation parameters was carried out using the high throughput Gene Pulser MXcell electroporation system. Just 4 hours post electroporation with a GAPDH siRNA, Jurkat cells expressed 88% less GAPDH mRNA than controls transfected with scramble siRNA. The same degree of silencing persisted at 24 and 48 hours post electroporation. Transfection of cells with fluorescently labeled siRNA resulted in 75% transfection efficiency for Neuro‐2A, 93% for HPF and 98% for HUVEC cells, as analyzed by flow cytometry. We provide optimized electroporation conditions for these and other difficult to transfect cells.

Nuclear localization signal peptides enhance transfection efficiency of chitosan/DNA complexes delivery

The purpose of this study was to investigate the effect of nuclear localization signal “KPKKKRKV” (NLS) on the enhancement of transfection efficiency of chitosan/DNA complexes delivery. The gene transfer efficiency of chitosan/DNA complexes with and without NLS was evaluated in a human cervical carcinoma cell line (Hela cells). The chitosan/DNA complex containing NLS increased transfection efficiencies in a NLS‐dose dependent manner on Hela cells, compared to control (chitosan/DNA complex or NLS). Cytotoxicity of the NLS/chitosan/DNA complexes increased as the amount of the peptide increased, however, over 80 % average cell viability was observed for complexes at the effective concentration of the peptide for transfections. Therefore, the NLS is expected to be a potent transfection enhancing agent without a covalent modification of pDNA or chitosan. Our findings suggest that the high gene expression with the negligible cytotoxicity can be achieved by adding the NLS peptide to chitosan/DNA complexes at optimal ratio. This research was supported by the Thailand Research Fund and Silpakorn University Research and Development Institute, Thailand.

Modest PGC-1α Overexpression in Muscle in Vivo Is Sufficient to Increase Insulin Sensitivity and Palmitate Oxidation in Subsarcolemmal, Not Intermyofibrillar, Mitochondria

PGC-1alpha overexpression in skeletal muscle, in vivo, has yielded disappointing and unexpected effects, including disrupted cellular integrity and insulin resistance. These unanticipated results may stem from an excessive PGC-1alpha overexpression in transgenic animals. Therefore, we examined the effects of a modest PGC-1alpha overexpression in a single rat muscle, in vivo, on fuel-handling proteins and insulin sensitivity. We also examined whether modest PGC-1alpha overexpression selectively targeted subsarcolemmal (SS) mitochondrial proteins and fatty acid oxidation, because SS mitochondria are metabolically more plastic than intermyofibrillar (IMF) mitochondria. Among metabolically heterogeneous rat hindlimb muscles, PGC-1alpha was highly correlated with their oxidative fiber content and with substrate transport proteins (GLUT4, FABPpm, and FAT/CD36) and mitochondrial proteins (COXIV and mTFA) but not with insulin-signaling proteins (phosphatidylinositol 3-kinase, IRS-1, and Akt2), nor with 5'-AMP-activated protein kinase, alpha2 subunit, and HSL. Transfection of PGC-1alpha into the red (RTA) and white tibialis anterior (WTA) compartments of the tibialis anterior muscle increased PGC-1alpha protein by 23-25%. This also induced the up-regulation of transport proteins (FAT/CD36, 35-195%; GLUT4, 20-32%) and 5'-AMP-activated protein kinase, alpha2 subunit (37-48%), but not other proteins (FABPpm, IRS-1, phosphatidylinositol 3-kinase, Akt2, and HSL). SS and IMF mitochondrial proteins were also up-regulated, including COXIV (15-75%), FAT/CD36 (17-30%), and mTFA (15-85%). PGC-1alpha overexpression also increased palmitate oxidation in SS (RTA, +116%; WTA, +40%) but not in IMF mitochondria, and increased insulin-stimulated phosphorylation of AKT2 (28-43%) and rates of glucose transport (RTA, +20%; WTA, +38%). Thus, in skeletal muscle in vivo, a modest PGC-1alpha overexpression up-regulated selected plasmalemmal and mitochondrial fuel-handling proteins, increased SS (not IMF) mitochondrial fatty acid oxidation, and improved insulin sensitivity.

Several Serum Proteins Significantly Decrease Inflammatory Response to Lipid-based Non-viral Vectors

Modifications of lipid-based gene delivery systems to improve efficacy is a continued effort in gene therapy research. Current advances include targeting specificity, controlled release and small molecule incorporation. While showing great progress, additional advances are necessary to increase transfection efficiency and decrease inflammatory toxicity. Previously, we demonstrated that sequentially injecting liposome, followed by DNA resulted in high transfection levels, but also significantly decreased the inflammatory response typically associated with lipoplex delivery. Here we attempt to elucidate the role of serum proteins in vector efficacy. Both lipoplexes and sequential complexes formed in the presence of mouse serum were examined by 2D gel electrophoresis. Unique or abundant proteins in sequential complexes were investigated for potential anti-inflammatory or target specific activity. Several serum proteins significantly reduced inflammation, while increasing the levels of transgene activity up to threefold compared to lipoplex. Furthermore, the same proteins did not decrease cytokine levels when added to preformed lipoplex. The results indicate sequential complexes (i.e., liposome mixed with protein, then DNA) and lipoplex are fundamentally different. These results suggest that a combination of protein content and DNA placement within the structure is responsible for the significantly improved efficacy and decreased inflammatory toxicity of these modified non-viral vectors.

Evaluation of the transfection property of a peptide ligand for the fibroblast growth factor receptor as part of PEGylated polyethylenimine polyplex

Purpose: Experiments were conducted to evaluate the utility of a peptide receptor ligand to improve transfection efficiency as part of a polyethylenimine-polyethylene glycol (PEI-PEG) polyplex. The 7-mer peptide (MQLPLAT), targeted toward the fibroblast growth factor 2 (FGF2) receptor, was recently identified using a phage-display library method as possessing a high degree of specificity for the FGF2 receptor without the mutagenicity associated with FGF itself. Two approaches (pre-modification or post-modification) to incorporate the peptide into the PEGylated polyplex were compared in terms of their effect on particle size, surface charge, DNA condensation ability, toxicity, cellular uptake and transfection efficiency.Methods: The peptide was conjugated to branched PEI (25 kDa) via a PEG spacer either before (pre-modified) or after (post-modified) complexation of PEI with DNA. Polyethyleneimine was conjugated to the PEG spacer (N-hydroxy succinimide (NHS) -PEG-maleimide (Mal)) through the NHS group. The FGF2 peptide was synthesized to contain a cysteine at the carboxyl end (MQLPLATC) and conjugated to the PEG spacer via the Maleimide group. Conjugates were evaluated using 1H NMR, amino acid analysis, and picrylsulfonic acid assay. DNA condensation was evaluated using agarose gel electrophoresis and cellular toxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cellular uptake was measured using flow cytometry and transfection efficiency was determined using a luciferase reporter gene assay.Results: Both pre- and post-modification approaches led to a decrease in the zeta potential of the resulting polyplexes but did not alter their size. The pre-modification of PEI did not affect its ability to condense DNA. However, polyplexes formed with the pre-conjugated PEI did not improve cell uptake or transfection efficiency. In contrast, polyplexes that were post-modified with the FGF2 peptide resulted in a 3-fold increase in cell uptake and a 6-fold increase in transfection efficiency. Both pre- and post-modified polyplexes resulted in lower toxicity compared with unmodified PEI.Conclusions: The results indicate that the FGF2 peptide improves transfection efficiency when used as part of post-modified PEI/PEG polyplex. When used with pre-modified PEI/PEG, the beneficial effect of the peptide on transfection is not evident, probably because, in this case, the peptide ligand is not readily accessible to the FGF receptor.

Topotecan and small interfering RNA suppress survivin expression synergistically in Caki-1 renal cancer cells: Direct suppression of survivin and enhancement of transfection efficiency by topotecan

Survivin is an apoptosis inhibitor found in many tumors but not in most normal differentiated tissues, which makes it an exciting target for cancer therapy. Survivin expression has been shown to be associated with cell proliferation in renal cancer and we previously demonstrated the possibility of treating renal cancer with survivin-specific siRNA and topotecan, which increased the uptake of siRNA by KU19-20 renal cancer cells. We found in the present study that the combination of siRNA and topotecan inhibits survivin expression in Caki-1 renal cancer cells by another mechanism. Caki-1 renal cancer cells expressed higher level of survivin than KU19-20 cells did and survivin-specific siRNA alone suppressed survivin expression only slightly. The combination of topotecan and siRNA suppressed survivin expression completely and inhibited cell proliferation. Topotecan itself did not increase cellular uptake of siRNA but suppressed survivin expression. It also increased transfection efficiency without increasing cellular uptake of siRNA. Although the mechanism of action varies between cell lines, combination therapy using topotecan and siRNA should offer an attractive approach for treatment of advanced renal cancer.

Synthesis and Characterization of Four-Arm Poly(ethylene glycol)-Based Gene Delivery Vehicles Coupled to Integrin and DNA-Binding Peptides

The goal of this study was to develop a gene delivery vehicle that can specifically target cell surface receptors with low nonspecific protein adsorption and low cytotoxicity. Toward this goal, four-arm poly(ethylene glycol) vehicles were functionalized with DNA-binding peptides (DBPs) and integrin-binding (RGD) peptides. We have previously described a novel PEG-based gene delivery vehicle functionalized with DBPs that successfully transfected Chinese hamster ovary (CHO) cells with low toxicity and low protein adsorption. This work investigated whether incorporating RGD peptides onto PEG-DBP vehicles could target specific cell surface receptors and increase transfection efficiency of HEPG2 cells. DBP and RGD peptides were coupled onto PEG-tetraacrylate (PEG-TA) in three combinations (molar ratios of DBP:RGD of 1:3, 2:2, and 3:1) and characterized by measuring particle size, zeta potential, and transfection efficiency as a function of charge ratio (peptide amine groups:DNA phosphate). Nonspecific protein adsorption and cytotoxicity of PEG-DBP-RGD vehicles were also measured. Dynamic light scattering showed that PEG-DBP-RGD vehicles condensed DNA into particles having mean diameters of 250-300 nm and zeta potentials ranging from -10 to 7 mV. It was found that coupling two RGD peptides to the PEG-DBP 2 vehicle increased the transfection efficiency at a polymer/DNA charge ratio of 5:1 (+/-) and 6:1 (+/-) and that these vehicles had transfection efficiencies similar to those of polyethylenimine (PEI)/DNA particles. However, coupling one or three RGD peptides to PEG-DBP vehicles did not increase the transfection efficiency. Additionally, the PEG-DBP-RGD/DNA particles adsorbed less protein than PEI particles and were less toxic to HEPG2 cells.

Low molecular weight hyaluronan shielding of DNA/PEI polyplexes facilitates CD44 receptor mediated uptake in human corneal epithelial cells

It was the aim of this study to prepare purified DNA/PEI polyplexes, which are coated with hyaluronan to facilitate CD44 receptor mediated uptake of the DNA/PEI polyplex and to reduce unspecific interactions of the complex with negatively charged extracellular matrix components on the ocular surface. Hyaluronans of different molecular weights (<10 kDa, 10-30 kDa and 30-50 kDa) were isolated after enzymatic degradation of high molecular weight hyaluronan via ultrafiltration by centrifugation. The influence of the different hyaluronans used for coating on the stability and transfection efficiency of the complexes was evaluated in vitro. Transfection and uptake studies were performed in human corneal epithelial (HCE) cells. CD44 receptor expression of this cell model was evaluated by immunohistochemistry. Coating of purified DNA/PEI polyplexes with low molecular weight hyaluronan (<10 kDa) facilitated receptor-mediated uptake via the CD44 receptor in HCE cells, increased complex stability in vitro, and effectively shielded the positive surface charges of the polyplex without decreasing its transfection efficiency. Higher molecular weights and larger amounts of hyaluronan in the complexes resulted in lesser improvements in the stability and transfection efficacy of the complexes. Coating of polyplexes with low molecular weight hyaluronan is a promising strategy for gene delivery to the ocular surface, where CD44 receptor mediated uptake decreased cytotoxicity and reduced non-specific interactions with the negatively charged extracellular matrix components are considered beneficial for increased transfection efficiency of non-viral vectors.