Using disaccharides to enhance in vitro and in vivo transgene expression mediated by a lipid-based gene delivery system

BACKGROUND: Lipid-based vectors have been widely applied to in vivo and in vitro gene delivery. Disaccharides can effectively stabilize lipid membranes. This study examined whether disaccharides could enhance the transgene expression mediated by lipid-based vectors. METHODS: Different disaccharides were incorporated into the vectors prepared with DOTAP/protamine/DNA (LPD) or with DNA/cationic liposomes containing DOTAP, DOTAP/Chol, DOTAP/DOPE, or DC-Chol/DOPE. The levels of transgene expression and internalized plasmid of CHO cells were represented by the percentages of GFP-positive cells and the fluorescence intensity of ethidium-monoazide covalently labeled plasmid, respectively. The vectors containing either cellobiose or trehalose were also intravenously injected into mouse tail vein to investigate the potentials of in vivo applications. RESULTS: For enhancing the transgene expression, cellobiose was found to be effective for all the vectors whereas maltose decreased the effectiveness of DOTAP/Chol liposomes and LPD. For the internalization of plasmid, most disaccharides were able to increase the cellular delivery of DOTAP, DOTAP/Chol, and DOTAP/DOPE liposomes, but caused decreases in the cellular entry of DC-Chol/DOPE liposomes. An approximately linear correlation between the internalized plasmid and the transgene expression was observed for all the treatments in this study. When the vectors were administered to mouse by intravenous injection, 10-fold and 3-fold increases in the luciferase expression of lung were observed for DOTAP liposomes containing 330 mM cellobiose and trehalose, respectively. CONCLUSIONS: This study showed that using trehalose and cellobiose with a lipid-based delivery system provides a straightforward approach to effectively enhance both in vitro and in vivo transgene expression.     

Adenoviral gene delivery to primary human cutaneous cells and burn wounds

The adenoviral transfer of therapeutic genes into epidermal and dermal cells is an interesting approach to treat skin diseases and to promote wound healing. The aim of this study was to assess the in vitro and in vivo transfection efficacy in skin and burn wounds after adenoviral gene delivery. Primary keratinocytes (HKC), fibroblasts (HFB), and HaCaT cells were transfected using different concentrations of an adenoviral construct (eGFP). Transfection efficiency and cytotoxicity was determined up to 30 days. Expression was quantified by FACS analysis and fluorimeter. Cytotoxicity was measured using the trypan blue exclusion method. 45 male Sprague Dawley rats received 2x10(8) pfu of Ad5-CMV-LacZ or carrier control intradermally into either superficial partial thickness scald burn or unburned skin. Animals were euthanized after 48 h, 7 or 14 days posttreatment. Transgene expression was assessed using immunohistochemistry and bioluminescent assays. The highest transfection rate was observed 48 h posttransfection: 79% for HKC, 70% for HFB, and 48% for HaCaT. The eGFP expression was detectable in all groups over 30 days (P>0.05). Cytotoxic effects of the adenoviral vector were observed for HFB after 10 days and HaCaT after 30 days. Reporter gene expression in vivo was significantly higher in burned skin compared with unburned skin (P=0,004). Gene expression decreases from 2 to 7 days with no significant expression after 14 days. This study demonstrates that effective adenoviral-mediated gene transfer of epidermal primary cells and cell-lines is feasible. Ex vivo gene transfer in epithelial cells might have promise for the use in severely burned patients who receive autologous keratinocyte sheets. Transient cutaneous gene delivery in burn wounds using adenoviral vectors causes significant concentrations in the wound tissue for at least 1 week. Based on these findings, we hypothesize that transient cutaneous adenoviral gene delivery of wound healing promoting factors has potential for clinical application.     

Chitosan enhanced gene delivery of cationic liposome via non-covalent conjugation

Two new types of stable ternary complexes were formed by mixing chitosan with DOTAP/pDNA lipoplex and DOTAP with chitosan/pDNA polyplex via non-covalent conjugation for the efficient delivery of plasmid DNA. They were characterized by atomic force microscopy, gel retarding, and dynamic light scattering. The DOTAP/CTS/pDNA complexes were in compacted spheroids and irregular lump of larger aggregates in structure, while the short rod- and toroid-like and donut shapes were found in CTS/DOTAP/pDNA complexes. The transfection efficiency of the lipopolyplexes showed higher GFP gene expression than DOTAP/pDNA and CTS/pDNA controls in Hep-2 and Hela cells, and luciferase gene expression 2–3-fold than DOTAP/pDNA control and 70–120-fold than CTS/pDNA control in Hep-2 cells. The intracellular trafficking was examined by confocal laser scanning microscopy. Rapid pDNA delivery to the nucleus enchanced by chitosan was achieved after 4 h transfection.     

Electroporation enhances reporter gene expression following delivery of naked plasmid DNA to the lung

BACKGROUND: Existing methods of non-viral airway gene transfer suffer from low levels of efficiency. Electroporation has been used to enhance gene transfer in a range of tissues. Here we assess the usefulness of electroporation for enhancing gene transfer in the lungs of mice and sheep. METHODS: Naked plasmid DNA (pDNA) expressing either luciferase or green fluorescent protein (GFP) was delivered to mouse lungs by instillation. Following surgical visualisation, the lungs were directly electroporated and the level and duration of luciferase activity was assessed and cell types that were positive for GFP were identified in lung cryosections. Naked pDNA was nebulised to the sheep lung and electrodes attached to the tip of a bronchoscope were used to electroporate airway segment bifurcations, Luciferase activity was assessed in electroporated and control non-electroporated regions, after 24 h. RESULTS: Following delivery of naked pDNA to the mouse lung, electroporation resulted in up to 400-fold higher luciferase activity than naked pDNA alone when luciferase was under the control of a cytomegalovirus (CMV) promoter. Following delivery of a plasmid containing the human polyubiquitin C (UbC) promoter, electroporation resulted in elevated luciferase activity for at least 28 days. Visualisation of GFP indicated that electroporation resulted in increased GFP detection compared with non-electroporated controls. In the sheep lung electroporation of defined sites in the airways resulted in luciferase activity 100-fold greater than naked pDNA alone. CONCLUSIONS: These results indicate that electroporation can be used to enhance gene transfer in the lungs of mice and sheep without compromising the duration of expression.     

Hydrodynamics-based procedure involves transient hyperpermeability in the hepatic cellular membrane: implication of a nonspecific process in efficient intracellular gene delivery

BACKGROUND: The mechanisms underlying the efficient gene transfer by a large-volume and high-speed intravenous injection of naked plasmid DNA (pDNA), a so-called hydrodynamics-based procedure, remain unclear and require further investigation. In this report, we have investigated possible mechanisms for the intracellular transport of naked pDNA by this procedure. METHODS: Propidium iodide (PI), a fluorescent indicator for cell membrane integrity, and luciferase- or green fluorescent protein (GFP)-expressing pDNA were injected into mice by the hydrodynamics-based procedure. RESULTS: PI was efficiently taken up by hepatocytes which appeared to be viable following the hydrodynamics-based procedure. Pre-expressed GFP in the cytosol was rapidly eliminated from the hepatocytes by a large-volume injection of saline. The profiles of plasma ALT and AST showed a steady decline with the highest values observed immediately after the hydrodynamics-based procedure. These results suggest that the hydrodynamics-based procedure produces a transient increase in the permeability of the cell membrane. The cellular uptake process appeared nonspecific, since simultaneous injection of an excess of empty vector did not affect the transgene expression. Sequential injections of a large volume of pDNA-free saline followed by naked pDNA in a normal volume revealed that the increase in membrane permeability was transient, with a return to normal conditions within 30 min. Transgene expression was observed in hepatocyte cultures isolated 10 min after pDNA delivery and in the liver as early as 10 min after luciferase-expressing RNA delivery, indicating that pDNA delivered immediately by the hydrodynamics-based procedure has the potential to produce successful transgene expression. CONCLUSIONS: These findings suggest that the mechanism for the hydrodynamics-based gene transfer would involve in part the direct cytosolic delivery of pDNA through the cell membrane due to transiently increased permeability.     

Successful genetic transduction in vivo into synovium by means of electroporation

This present study aims at establishing a novel in vivo gene delivery system for intra-articular tissues. Plasmid DNA (pDNA) carrying the firefly luciferase or enhanced green fluorescent protein (EGFP) genes as markers was injected into a joint space and electric stimuli were given percutaneously with a pair of electrodes. Injection with naked pDNA alone did not induce any detectable level of luciferase activity, whereas electroporation at 25-500 V/0.7 cm resulted in a significant expression of the marker gene in the synovium. The expression level depended on the voltage, the optimum transfection being achieved at 150 V/0.7 cm. When the Epstein-Barr virus (EBV)-based plasmid vectors harboring the EBV nuclear antigen 1 (EBNA1) gene and oriP sequence were substituted for conventional pDNA, the transfection efficiency was increased approximately 5-10 times. Histological examination of the EGFP gene-transfected joints revealed that the marker gene was expressed in the synovial membrane while other intra-articular tissues such as articular cartilage were negative for the transgene product. Transgene-specific mRNA was demonstrated in synovium but not in other organs as estimated by RT-PCR analysis. The present results strongly suggest that in vivo electroporation is a quite simple, safe, and effective gene delivery method that could be applicable to gene therapy against articular diseases.     

Stimulatory effect of polyethylene glycol (PEG) on gene expression in mouse liver following hydrodynamics-based transfection

BACKGROUND: Rapid intravenous injection of a large volume of plasmid DNA (pDNA), i.e. a transfection procedure based on hydrodynamics, is known to be an efficient and liver-specific method of in vivo gene delivery. However, the gene expression is transient. METHODS: We investigated the effect of addition of polyethylene glycol (PEG) to a solution of naked pDNA (luciferase) on the expression of the gene in mouse liver following transfection by the hydrodynamics-based technique. In addition, the mechanism leading to the enhancement of the gene expression was studied. RESULTS: The addition of 1% (w/v) PEG2000 to the pDNA solution enhanced the resulting gene expression in the liver. Increasing the PEG2000 concentration to more than 1 and up to 10% (w/v) rather diminished the gene expression level. By contrast, increasing the molecular weight of PEG to over 2000 up to 10 000 did not affect the level of gene expression. Histopathological and serum-chemistry examinations indicated that hydrostatic or osmotic pressure increased tissue and hepatocellular damage in a PEG-concentration-dependent manner, and resulted in a decrease in gene expression. Quantitative evaluation showed that the enhanced gene expression resulted from stabilization of the pDNA introduced into the hepatocytes and an enhancement of the transport of intact pDNA to the nucleus. CONCLUSIONS: For most gene therapy applications and gene function studies, sustained expression of the introduced gene(s) is necessary. This simple method to achieve enhanced gene expression in liver may have a great potential for a wide variety of laboratory studies in molecular and cellular biology as well as possibly for future clinical applications in humans.     

Polybrene improves transfection efficacy of recombinant replication-deficient adenovirus in cutaneous cells and burned skin

BACKGROUND: The hostile environment found in acute and chronic wounds decreases the physiological half-life of purified synthetic or recombinant peptides dramatically. Gene therapy, on the other hand, may be a viable option since it relies on the cellular machinery of the host to locally manufacture the proteins of interest. The aim of this study was to evaluate and optimize the local administration of transient cutaneous adenoviral gene delivery in wounds. METHODS: Primary human keratinocytes (HKC) and HaCaT cells were transfected with replication-deficient adenovirus (Ad5) containing the reporter gene for beta-galactosidase (LacZ). The vector was used alone or precoated with either (1) Lipofectamine 2000, (2) FuGENE 6, or (3) Polybrene. For in vivo testing a rat burn model was used. Animals were randomized into three groups: (1) Ad5-LacZ alone; (2) Ad5-LacZ precoated with Polybrene, or (3) carrier control (phosphate-buffered saline (PBS)). Samples were harvested from burned and unburned tissue sections after either 48 h or 7 days. Transgene expression was quantified by bioluminometric assay and localized using immunohistochemistry. A BrdU assay was performed to determine the influence of the used transfection reagents on cell proliferation. RESULTS: Transfection efficacy was significantly improved in vitro (p < 0.001) as well as in partial thickness burned (p = 0.015) and unburned skin (p > 0.001) after precoating Ad5 with Polybrene compared to Ad5 alone. Transgene expression was 10-fold higher in burned skin (9305 pg/mg protein) compared to unburned skin (859 pg/mg protein). CONCLUSIONS: It is feasible to improve transfection efficacy in vitro and in vivo by precoating the adenovirus with Polybrene.     

阳离子脂质体介导基因转染肿瘤细胞

使用基因转运载体运载肿瘤细胞进行转染是基因治疗的关键环节之一。Lipo-fectamine2000和DOTAP作为商品转染试剂,具有较高的转染效率。为了进一步发掘其作为基因转运载体的应用潜力,该文研究了Lipofectamine2000和DOTAP的粒径、Zeta电位及形态,并分别与绿色荧光蛋白基因（pGFP—N2）、荧光素酶基因（pGL3）结合,形成脂质体／DNA复合物,通过载入人喉癌细胞（Hep-2）和人肺癌细胞（NCI—H460）,考察了其转染效率和细胞毒性。结果表明,脂质体Lipofectamine2000与DOTAP都能有效压缩DNA,形成复合物。Lipofectamine2000与DOTAP井目比,转染效率高,与DNA最佳转染比例范围为2：1～4：1。毒性实验显示,在N／P大于3／l时,Lipofectamine2000与DOTAP对癌细胞具有一定的细胞毒性。细胞种类对脂质体的转染效率有很大影响,Lipo—fectamine2000对Hep－2细胞的转染效率比NcI—H460高。     

Cationic Liposomes Containing Disulfide Bonds in Delivery of Plasmid DNA

Abstract

Cationic liposomes are non-viral gene transfer vectors for in vitro and in vivo experiments. In the present studies, we investigated whether a disulfide linkage in a cationic lipid was reducible by cell lysate resulting in the release of plasmid DNA and enhanced gene transfection. We also investigated if the differences in transgene production were from differences in total amount of cellular associated plasmid DNA. We systematically compared the gene transfection of disulfide bond containing-cationic lipid, 1', 2'-dioleoyl-sn-glycero-3'-succinyl-2-hydroxyethyl disulfide ornithine conjugate (DOGSDSO), its non-disulfide-containing analog, 1', 2'-dioleyl-sn-glycero-3'-succinyl-1, 6-hexanediol ornithine conjugate (DOGSHDO), 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP). Two transgene reporter systems (i.e., luciferase and green fluorescent protein (GFP)) were used to address transgene transgene expression and transgene efficiency. Experiments with the luciferase expression plasmid resulted in transgene activity up to 11 times greater transgene production for the disulfide containing lipid in at least two different cell lines, COS 1 and CHO cells. When transgene expression was determined by GFP activity, DOGSDSO liposomes were four times greater than the non-disulfide lipid or positive control (DOTAP) liposomes. By quantifying nucleic acid uptake by flow cytometry it was also demonstrated that increase expression was not solely from an increase in cellular plasmid DNA accumulation. These results demonstrate that cationic lipids containing a disulfide linkage are a promising method for gene transfer.     

In vivo Site-Specific Transfection of Naked Plasmid DNA and siRNAs in Mice by Using a Tissue Suction Device

We have developed an in vivo transfection method for naked plasmid DNA (pDNA) and siRNA in mice by using a tissue suction device. The target tissue was suctioned by a device made of polydimethylsiloxane (PDMS) following the intravenous injection of naked pDNA or siRNA. Transfection of pDNA encoding luciferase was achieved by the suction of the kidney, liver, spleen, and heart, but not the duodenum, skeletal muscle, or stomach. Luciferase expression was specifically observed at the suctioned region of the tissue, and the highest luciferase expression was detected at the surface of the tissue (0.12±0.03 ng/mg protein in mice liver). Luciferase expression levels in the whole liver increased linearly with an increase in the number of times the liver was suctioned. Transfection of siRNA targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene significantly suppressed the expression of GAPDH mRNA in the liver. Histological analysis shows that severe damage was not observed in the suctioned livers. Since the suction device can be mounted onto the head of the endoscope, this method is a minimally invasive. These results indicate that the in vivo transfection method developed in this study will be a viable approach for biological research and therapies using nucleic acids.     

Plasmid DNA-entrapped nanoparticles engineered from microemulsion precursors: in vitro and in vivo evaluation

Nonviral gene therapy has been a rapidly growing field. However, delivery systems that can provide protection for pDNA and potential targeting are still desired. A novel pDNA-nanoparticle delivery system was developed by entrapping hydrophobized pDNA inside nanoparticles engineered from oil-in-water (O/W) microemulsion precursors. Plasmid DNA was hydrophobized by complexing with cationic surfactants DOTAP and DDAB. Warm O/W microemulsions were prepared at 50-55 degrees C with emulsifying wax, Brij 78, Tween 20, and Tween 80. Nanoparticles were engineered by simply cooling the O/W microemulsions containing the hydrophobized pDNA in the oil phase to room temperature while stirring. The nanoparticles were characterized by particle sizing, zeta-potential, and TEM. Nanoparticles were challenged with serum nucleases to assess pDNA stability. In addition, the nanoparticles were coincubated with simulated biological media to assess their stability. In vitro hepatocyte transfection studies were completed with uncoated nanoparticles or nanoparticles coated with pullulan, a hepatocyte targeting ligand. In vivo biodistribution of the nanoparticles containing I-125 labeled pDNA was monitored 30 min after tail-vein injection to Balb/C mice. Depending on the hydrophobizing lipid agent employed, uniform pDNA-entrapped nanoparticles (100-160 nm in diameter) were engineered within minutes from warm O/W microemulsion precursors. The nanoparticles were negatively charged (-6 to -15 mV) and spherical. An anionic exchange column was used to separate unentrapped pDNA from nanoparticles. Gel permeation chromatography of pDNA-entrapped and serum-digested nanoparticles showed that the incorporation efficiency was approximately 30%. Free 'naked' pDNA was completely digested by serum nucleases while the entrapped pDNA remained intact. Moreover, in vitro transfection studies in Hep G2 cells showed that pullulan-coated nanoparticles resulted in enhanced luciferase expression, compared to both pDNA alone and uncoated nanoparticles. Preincubation of the cells with free pullulan inhibited the transfection. Finally, 30 min after tail vein injection to mice, only 16% of the 'naked' pDNA remained in the circulating blood compared to over 40% of the entrapped pDNA. Due to the apparent stability of these pDNA-entrapped nanoparticles in the blood, they may have potential for systemic gene therapy applications requiring cell and/or tissue-specific delivery.     

Proteolipidic vectors for gene transfer to the lung

In order to develop improved synthetic gene transfer vectors, we have synthesized bifunctional peptides composed of a DNA binding peptide (P2) and ligand peptides selected by the phage display technique on tracheal epithelial cells. We have evaluated the capacity of these peptides to enhance the gene transfer efficiency of the cationic lipid DOTAP to the mouse lung. To optimize the in vivo transfection efficiency, we first compared the efficiency of DOTAP to transfect the lung by either intravenous injection or aerosolization. We then tested DNA/Peptide/DOTAP complexes formed at different Peptide/DNA and DOTAP/DNA charge ratios. Under optimal conditions, precompaction of DNA by peptide P2 gave a higher expression in the mouse lung using the luciferase reporter gene than DOTAP/DNA complexes. A further increase of transfection efficiency was obtained with the bifunctional peptide P2-9. Experiments performed with the GFP reporter gene showed expression in the alveolar parenchyme.     

Nanoparticles of compacted DNA transfect postmitotic cells

Charge-neutral DNA nanoparticles have been developed in which single molecules of DNA are compacted to their minimal possible size. We speculated that the small size of these DNA nanoparticles may facilitate gene transfer in postmitotic cells, permitting nuclear uptake across the 25-nm nuclear membrane pore. To determine whether DNA nanoparticles can transfect nondividing cells, growth-arrested neuroblastoma and hepatoma cells were transfected with DNA/liposome mixtures encoding luciferase. In both models, growth-arrested cells were robustly transfected by compacted DNA (6,900-360-fold more than naked DNA). To evaluate mechanisms responsible for enhanced transfection, HuH-7 cells were microinjected with naked or compacted plasmids encoding enhanced green fluorescent protein. Cytoplasmic microinjection of DNA nanoparticles generated a approximately 10-fold improvement in transgene expression as compared with naked DNA; this enhancement was reversed by the nuclear pore inhibitor, wheat germ agglutinin. To determine the upper size limit for gene transfer, DNA nanoparticles of various sizes were microinjected into the cytoplasm. A marked decrease in transgene expression was observed as the minor ellipsoidal diameter approached 25 nm. In summary, suitably sized DNA nanoparticles productively transfect growth arrested cells by traversing the nuclear membrane pore.     

Protamine Sulfate Provides Enhanced and Reproducible Intravenous Gene Transfer by Cationic Liposome/DNA Complex

Abstract

A novel lipid/polycation/DNA (LPD) formulation has been developed for in vivo gene transfer. It involves the condensation of plasmid DNA with protamine sulfate, a cationic polypeptide, followed by the addition of DOTAP cationic liposomes. Compared with DOTAP/DNA complex, LPD offers greater protection of plasmid DNA against enzymatic digestion and gives consistently higher gene expression in mice via tail vein injection. The in vivo efficiency of LPD was dependent upon charge ratio and was also affected by the lipid used. Increasing the amount of DNA delivered induced an increase in gene expression. The optimal dose was approximately 50 μg per mouse, at which concentration approximately 10 ng luciferase protein per mg extracted tissue protein could be detected in the lung. Gene expression in the lung was detected as early as 1 h after injection, peaked at 6 h, and declined thereafter. Using LacZ as a reporter gene, it was shown that endothelial cells were the primary locus of transgene expression in both lung and spleen. No sign of inflammation in these organs was noticed. Since protamine sulfate has been proven to be non-toxic and only weakly immunogenic in humans, this novel vector may be useful for the clinical use of gene therapy.     

Transfection property of a new cholesterol-based cationic lipid containing tri-2-hydroxyethylamine as gene delivery vehicle

A novel cholesterol-based cationic lipid containing a tri-2- hydroxyethylamine head group and ether linker (Chol- THEA) was synthesized and examined as a potent gene delivery vehicle. In the preparation of cationic liposome, the addition of DOPE as helper lipid significantly increased the transfection efficiency. To find the optimum transfection efficiency, we screened various weight ratios of DOPE and liposome/DNA (N/P). The best transfection efficiency was found at the Chol-THEA:DOPE weight ratio of 1:1 and N/P weight ratio of 10~15. Most of the plasmid DNA was retarded by this liposome at the optimum N/P weight ratio of 10. The transfection efficiency of Chol-THEA liposome was compared with DOTAP, Lipofectamine, and DMRIE-C using the luciferase assay and GFP expression. Chol-THEA liposome with low toxicity had better or similar potency of gene delivery compared with commercial liposomes in COS-7, Huh-7, and MCF-7 cells. Therefore, Chol-THEA could be a useful non-viral vector for gene delivery.     

New methodologies to characterize the effectiveness of the gene transfer mediated by DNA-chitosan nanoparticles

In this work three DNA-chitosan nanoparticle formulations (Np), differing in the molecular weight (MW; 150 kDa, 400 kDa, and 600 kDa) of the polysaccharide, were prepared and administered by two different administration routes: the hydrodynamics-based procedure and the intraduodenal injection. After the hydrodynamic injection, DNA-chitosan nanoparticles were predominantly accumulated in the liver, where the transgene was expressed during at least 105 days. No significant influence of MW was observed on the levels of luciferase expression. The curves of bioluminescence versus time obtained using the charge-coupled device (CCD) camera were described and divided in three phases: (i) the initial phase, (ii) the sustained release step and (iii) the decline phase (promotor inactivation, immunological and physiological processes). From these curves, which describe the transgene expression profile, the behavior of the different formulations as gene delivery systems was characterized. Therefore, the following parameters such as C_max (maximum level of detected bioluminescence), AUC (area under the bioluminescence-time curve) and MET (mean time of the transgene expression) were calculated. This approach offers the possibility of studying and comparing transgene expression kinetics among a wide variety of gene delivery systems. Finally, the intraduodenal administration of naked DNA permitted the gene transfer in a dose dependent manner quantifiable with the CCD camera within 3 days. Nevertheless, the same administration procedure of the three formulations did not improve the levels of transgene expression obtained with naked DNA. This fact could be explained by the rapid physiological turn-over of enterocytes and by the ability of chitosan nanoparticles to control the DNA release.     

High volume hydrodynamic injection of plasmid DNA via the hepatic artery results in a high level of gene expression in rat hepatocellular carcinoma induced by diethylnitrosamine

BACKGROUND: Hydrodynamic injection of naked plasmid DNA (pDNA) via the tail vein is a safe and effective method of gene transfer to the liver. However, successful gene transfer has yet to be shown for hepatocellular carcinoma (HCC); therefore, we investigated the feasibility and efficacy of hydrodynamic injection via the tail vein and hepatic artery in a diethylnitrosamine (DEN)-induced HCC model in rats. METHODS: HCC was induced in Sprague-Dawley rats by 100 ppm DEN in drinking water. pCMV-SPORT-beta-galactosidase (beta-gal, 400 microg) was injected (i) via the tail vein in a volume of 0.1 ml/g in 30 s or (ii) via the hepatic artery in a volume of 5 or 10 ml at 1 ml/s, either with or without temporary occlusion of the inferior vena cava (IVC) and portal vein (PV). The liver was harvested 24 h after administration, and beta-gal expression was evaluated with X-gal staining and measurement of enzymatic activity in tissue homogenates. RESULTS: Hydrodynamic injection via the tail vein achieved transgene expression only in non-cancerous tissue (tumor: 0.16 +/- 0.04%, non-tumor: 5.07 +/- 1.66%). Hydrodynamic injection via the hepatic artery was tolerated, but failed to produce efficient transgene expression in tumor and non-tumor cells. On the other hand, concomitant use of temporary IVC/PV occlusion with hydrodynamic injection via the hepatic artery dramatically increased transgene expression in cancer cells, but tumor-selective gene transfer was not achieved with this procedure (tumor: 7.38 +/- 3.66%, non-tumor: 7.77 +/- 1.06%). CONCLUSIONS: High-volume hydrodynamic injection of a pDNA solution via the hepatic artery with IVC/PV occlusion achieved a high level of gene expression in a HCC rat model. This gene transfer technique may have potential in clinical gene therapy for HCC.     

Advanced transfection with Lipofectamine 2000 reagent: primary neurons, siRNA, and high-throughput applications

Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a simple protocol. Optimum transfection efficiency and subsequent cell viability depend on a number of experimental variables such as cell density, liposome and DNA concentrations, liposome-DNA complexing time, and the presence or absence of media components such as antibiotics and serum. The importance of these factors in Lipofectamine 2000 mediated transfection will be discussed together with some specific applications: transfection of primary neurons, high throughput transfection, and delivery of small interfering RNAs.