The third helix of the murine Hoxc8 homeodomain facilitates protein transduction in mammalian cells

Previously, we have demonstrated that purified Hoxc8 homeoprotein has the ability to penetrate the cellular membrane and can be transduced efficiently into COS-7 cells. Moreover, the Hoxc8 protein is able to form a complex with DNA molecules in vitro and helps the DNA be delivered intracellularly, serving as a gene delivery vehicle. Here, we further analyzed the membrane transduction activity of Hoxc8 protein and provide the evidence that the 16 amino acid (a.a.191-206, 2.23 kDa) third helix of murine Hoxc8 protein is an efficient protein transduction domain (PTD). When the 16 amino acid peptide was fused at the carboxyl terminal of enhanced green fluorescence protein (EGFP), the fusion proteins were transduced efficiently into the primary pig fetal fibroblast cells. The transduction efficiency increased in a concentration-dependent manner up to 1 μM, and appeared to plateau above a concentration of 1 μM. When tandem multimers of PTD, EGFP-PTD(2), EGFP-PTD(3), EGFP-PTD(4), and EGFP-PTD(5), were analyzed at 500 nM of concentration, the penetrating efficiency increased in a dose-dependent manner. As the number of PTDs increased, the EGFP signal also increased, although the signal maintained plateau after EGFP-PTD(3). These results indicate that the 16 amino acid third helix is the key element responsible for the membrane transduction activity of Hoxc8 proteins, and further suggest that the small peptide could serve as a therapeutic delivery vehicle for large cargo proteins.     

Efficient cellular uptake of recombinant murine Hoxc8 homeoprotein in COS-7 cells

In order to analyze the self-delivery activity of Hoxc8, recombinant Hoxc8 protein (rHoxc8) was designed to be expressed and purified in E. coli as a glutathione S-transferase and green fluorescent protein-fused form (GST-GFP-Hoxc8). After purification using glutathione sepharose beads, the 82 kDa fusion protein was separated on the SDS-PAGE gel and confirmed by detecting the fluorescence through luminescent image analyzer. When rHoxc8 was added to culture media for 30 h, most of the COS-7 cells contained the fusion proteins, showing green fluorescence under the fluorescent microscope. When the efficiency of cellular uptake was examined after Hoechst staining, almost 100% of the cells exhibited the GFP signal, revealing that rHoxc8 can traverse the cellular membrane of COS-7 cells efficiently, suggesting that the rHoxc8 could be applied in the development of efficient and useful delivery vectors for therapeutic molecules.     

Comparison of protein transduction domains in mediating cell delivery of a secreted CRE protein

Protein transduction domains (PTDs) are versatile peptide sequences that facilitate cell delivery of several cargo molecules including proteins. PTDs usually consist of short stretches of basic amino acids that can cross the plasma membrane and gain entry into cells. Traditionally, to assess PTD mediated protein delivery, PTD-fusion proteins have been used as purified proteins. To overcome the requirement for a protein purification step, we used a secretory signal peptide to allow PTD-CRE fusion proteins to be exported from transfected mammalian cells. PTD induced protein transduction into cells was assessed by a CRE-mediated recombination event that resulted in beta-galactosidase expression. Several PTDs were tested including the prototypic TAT, different TAT variants, Antp, MTS and polyarginine. A negative correlation was observed between the cationic charge on the PTD and the extent of secretion. Poor secretion was found when the PTD charge was greater than +5. One TAT-CRE protein variant had a 14-fold enhancement above CRE alone when added to cells in the presence of chloroquine. This PTD domain also enhanced gene expression after plasmid delivery. These data illustrate that some secreted PTD proteins may be useful reagents to improve protein delivery in mammalian systems and a novel approach to enhancing the response to DNA transfections.     

A smart nanoassembly consisting of acid-labile vinyl ether PEG-DOPE and protamine for gene delivery: preparation and in vitro transfection

The conception of a modular designed and viruslike nonviral vector has been presented for gene delivery. Recently, we constructed a new smart nanoassembly (SNA) with multifunctional components that was composed of a condensed core of pDNA with protamine sulfate (PS) and a dioleoyl phosphatidylethanolamine (DOPE)-based lipid envelope containing poly(ethylene glycol)--vinyl ether--DOPE (PVD). SNAs with mPEG 2000 (SNAs1) or mPEG 5000 (SNAs2) loading PS/DNA were prepared by the lipid film hydration technique. The particle size was about 160 nm for SNAs1 and 240 nm for SNAs2 loading PS/DNA (10:1 w/w), and the zeta potential was about 4 mV for two SNAs. The in vitro release experiment indicated that PVD possessed a good ability for self-dePEGylation, which could result in the recovery of an excellent fusogenic capacity of DOPE at low pH. SNAs showed a higher transfection efficiency and much lower cytotoxicity than did Lipofectamine 2000 on HEK 293, HeLa, and COS-7 cells. The cellular uptake and subcellular localization demonstrated that the superior transfection efficiency of SNAs could result from the fact that the DOPE-based lipid envelope containing PVD increased PS/DNA in the cytoplasm, and protamine enhanced the nuclear delivery or overcame the nuclear membrane barrier. These results implied that the PVD-based nanoassembly loading PS/DNA could be a promising gene delivery system.     

Taking the cell by stealth or storm? Protein transduction domains (PTDs) as versatile vectors for delivery

A cell delivery system is increasing in use in many areas of cell and molecular biology and bio-medicine. This system is based on a number of naturally occurring protein motifs and/or sequences which show the remarkable ability to rapidly cross the mammalian cell membrane without compromising its structure or function. These so-called Protein Transduction Domains (PTDs) offer unprecedented advantages for intracellular delivery. These advantages include, but are not limited to, their applicability to all cell types (no cell type has yet been described which is not transduced by these PTDs), and the range of cargoes that can be transduced (including peptides, small proteins, full-length enzymes, DNA oligomers, peptide-nucleic acid oligomers, liposomes, and magnetic nanoparticles). Furthermore, the PTDs have been demonstrated to be suitable for in vivo delivery including delivery across the blood brain barrier, and have been shown to cross the plasma membrane rapidly and enter the cytoplasm and nuclear regions of the cell. In this review, the general properties of the most commonly used PTDs are described. The strategies currently being undertaken also highlight that improvements in membrane transduction are possible despite our lack of understanding of the exact biochemical and/or physical mechanisms of transduction. Recent examples of the range of potential applications are also discussed.     

Breaching biological barriers: protein translocation domains as tools for molecular imaging and therapy

The lipid bilayer of a cell presents a significant barrier for the delivery of many molecular imaging reagents into cells at target sites in the body. Protein translocation domains (PTDs) are peptides that breach this barrier. Conjugation of PTDs to imaging agents can be utilized to facilitate the delivery of these agents through the cell wall, and in some cases, into the cell nucleus, and have potential for in vitro and in vivo applications. PTD imaging conjugates have included small molecules, peptides, proteins, DNA, metal chelates, and magnetic nanoparticles. The full potential of the use of PTDs in novel in vivo molecular probes is currently under investigation. Cells have been labeled in culture using magnetic nanoparticles derivatized with a PTD and monitored in vivo to assess trafficking patterns relative to cells expressing a target antigen. In vivo imaging of PTD-mediated gene transfer to cells of the skin has been demonstrated in living animals. Here we review several natural and synthetic PTDs that have evolved in the quest for easier translocation across biological barriers and the application of these peptide domains to in vivo delivery of imaging agents.     

Biological applications of protein transduction technology

The impermeable nature of the cell membrane to peptides, proteins, DNA and oligonucleotides limits the therapeutic potential of these biological agents. However, the recent discovery of short cationic peptides that cross the plasma membrane efficiently is opening up new possibilities for the intracellular delivery of such agents. These peptides are commonly referred to as protein transduction domains (PTDs) and are successfully used to transport heterologous proteins, peptides and other types of cargo into cells. Several recent reports have used the membrane transducing technology in vivo to deliver biologically active cargo into various tissues. This review discusses the structure of the most commonly used PTDs and how their ability to transduce membranes is used to regulate biological functions. It also considers future directions and the potential of this technology to move from the laboratory into the clinic.     

脂质体DC-Chol/DOPE对HEK293FT细胞的高效转染及其在腺病毒制备中的应用

重组病毒载体系统因为具有高效的基因转移能力得到了广泛应用,而病毒包装细胞的转染是重组病毒制备过程中的关键步骤。优化了脂质体DC-Chol/DOPE介导的转染常用的病毒包装细胞系HEK293FT的实验条件,比较了DC-Chol/DOPE、Lipofectamine2000和磷酸钙共沉淀法转染细胞的效率,并且比较了用DC-Chol/DOPE和磷酸钙共沉淀法转染293FT细胞制备重组腺病毒的结果,发现DC-Chol/DOPE对293FT细胞的转染效率以及最终收获的病毒滴度都远高于磷酸钙共沉淀法转染。所以,利用DC-Chol/DOPE转染293FT细胞制备重组病毒是一种简单、高效、成本低廉的方法。     

Long duration electroporation for achieving high level expression of glucocorticoid receptors in mammalian cell lines

A method is presented that utilizes long duration electroporation (LDE) to more efficiently introduce DNA into mammalian cell lines than standard electroporation techniques. With SV40-based vectors, more than 550,000 glucocorticoid receptors (GRs) per cell could be obtained in COS-7 cells with good cell survival. In experiments with a CMV-driven vector expressing an enhanced Green Fluorescent Protein (EGFP), 54% of the cells were transfected, and 77% of EGFP positive cells expressed EGFP at moderate to high levels. In cell lines not containing the large T antigen, a CMV-driven vector for the GR was superior to the SV40-based vector. In EDR3, DG44, and CV-1 cell lines approximately 220,000, 190,000 and 150,000 GRs/cell were obtained, respectively. Transfection efficiency of the EGFP vector ranged from 44 to 55% for the three cell lines. Cortisol treatment of COS-7 and DG44 cultures cotransfected with vectors expressing the GR and a GRE driven luciferase gene produced 4 to 12 times more enzyme activity per plate with LDE than conventional electroporation protocols. LDE allows transient overexpression of proteins in COS-7 cells at the high levels generally achieved by mammalian overexpression systems only in stable cell lines.     

Evaluation of cationic assemblies constructed with amino acid based lipids for plasmid DNA delivery

We synthesized cationic lipids bearing lysine, histidine, or arginine as a cationic headgroup for use in gene transfer studies. The cationic assemblies formed from lysine- or arginine-type lipids gave unilamellar vesicles (approximately 100 nm diameter), whereas the morphology of the histidine-type lipids was tube-like. The competences of the cationic assemblies were sufficient to form lipoplexes, and the resulting lipoplexes were evaluated in terms of gene expression efficiencies with COS-7 cells. The lysine- or arginine-type lipids exhibited higher gene expression efficiencies than that of Lipofectamine2000, a conventional transgenic reagent, indicating that stable lipoplexes could be prepared between spherical cationic assemblies and plasmid DNA. The gene expression efficiency in relation to the cationic headgroup of the lipids was as follows: lysine > or = arginine > histidine. In addition, gene expression efficiency was enhanced by decreasing the length of the alkyl chain of the hydrophobic moiety. Unlike Lipofectamine2000, no reduction in transfection efficiency in the presence of fetal bovine serum was observed for the lipoplexes formed using synthetic cationic lipids. Moreover, the synthetic cationic lipids revealed remarkably low cytotoxicity compared with Lipofectamine2000. In conclusion, cationic assemblies formed from 1,5-ditetradecyl-N-lysyl-L-glutamate or 1,5-ditetradecyl-N-arginyl-L-glutamate can be used as an effective plasmid DNA delivery system.     

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.     

新型聚乙烯亚胺衍生物在COS-7细胞中转染和毒性的研究

目的：研究以乙二醛为连接剂的聚乙烯亚胺（Polyethyleneimine,PEI）衍生物Polyimine-PEI对非洲绿猴肾癌细胞COS-7的转染活性和细胞毒性的影响。方法：以荧光素酶质粒为报告基因,研究高分子与DNA的复合物在COS-7细胞的转染活性,用MTT方法研究高分子对COS-7细胞的毒性。结果：COS-7细胞实验显示,Polyimine-PEI具有很低细胞毒性,其毒性显著低于PEI25kDa,同时也具有高效输送质粒的能力。结论：Polyimine-PEI是一种新型的高效,低毒在基因治疗领域有相当前景的非病毒载体。     

Preparation, characterization and transfection efficacy of chitosan nanoparticles containing the intestinal trefoil factor gene

Intestinal trefoil factor (ITF) is a novel polypeptide with potential pharmacological value for the prevention and healing of tissue injury; however, poor production capacity limits its clinical application. Chitosan, as a non-viral vehicle, has been successfully used in gene delivery for its intrinsic characteristics. In this context, we prepared chitosan nanoparticles enwrapping ITF cDNA and investigated its size, zeta potential, stability, release profiles, loading efficiency and loading capacity. Gene transfer capability was assessed in HEK293 cells. The data revealed that the chitosan/DNA nanoparticles were successfully prepared with sizes less than 500 nm and positive zeta potentials. The nanoparticles could protect DNA from nuclease degradation, and release profiles of DNA were dependent on N/P ratios. In addition, transfection efficiency of chitosan/DNA nanoparticles was equivalent to Lipofectamine (TM). Collectively, the results suggest that chitosan/DNA nanoparticles could be a promising method for ITF gene therapy.     

Bead transfection of adherent cells

Bead transfection is a simple, rapid, efficient, and cost-effective method of gene transfer into adherent mammalian cells. It involves a brief incubation of the cells with glass beads in a solution containing the DNA to be transferred. We have optimized this technique using COS-7 (an SV40 transformed monkey kidney cell line) and a transient expression assay for chloramphenicol acetyl transferase (CAT). Stable transfection efficiency assessed using the selectable marker gene neomycin phosphotransferase (NEO^R) was 27% in COS-7 cells. As this technique delivers high transfection efficiency with little manipulation of the exogenous DNA and does not require the use of any viral sequences, it may be a useful alternative method of gene delivery in the development of gene therapy protocols.     

Expression of endostatin mediated by a novel non-viral delivery system inhibits human umbilical vein endothelial cells in vitro

Ultrasound (US)-mediated microbubble destruction is recognized to have considerable potential for gene delivery, whereas, there is few report of its effect on enhancing liposomal transfection. In this study, we used pIRES2-EGFP/hES containing human endostatin (hES) cDNA as target gene to test the hypothesis that US exposure with microbubbles could improve liposomal transfection, and to investigate the possibility of intracellular delivery of ES gene using this method. Under the controlled US exposure condition with microbubbles, the plasmid:liposome was transferred into COS-7 cells. The transfection rate, the expression of endostatin and the inhibition effect of transfection-endostatin on endothelial cells were assessed. The results revealed that US-mediated microbubble destruction together with liposome could significantly enhance gene transfection without obvious cell damage. By this means, endostatin gene could be efficiently transferred into COS-7 cells and expressed. The transfection-endostatin could inhibit endothelial proliferation and migration, which suggests that the non-viral method might be useful in anti-angiogenesis therapy in the future.     

Transient non-viral cutaneous gene delivery in burn wounds

BACKGROUND: Gene transfer to burn wounds could present an alternative to conventional and often insufficient topical and systemic application of therapeutic agents to aid in wound healing. The goals of this study were to assess and optimize the potential of transient non-viral gene delivery to burn wounds. METHODS: HaCaT cells were transfected with luciferase or beta-galactosidase transgene using either pure plasmid DNA (pDNA) or complexed with Lipofectamine 2000, FuGENE6, or DOTAP-Chol. Expression was determined by bioluminescence and fluorescence. Forty male Sprague-Dawley rats received naked pDNA, lipoplexes, or carrier control intradermally into either unburned skin, superficial, partial, or full-thickness scald burn. Animals were sacrificed after 24 h, 48 h, or 7 days, and transgene expression was assessed. RESULTS: Gene transfer to HaCaT cells showed the overall highest expression for DOTAP/Chol (77.85 ng luciferase/mg protein), followed by Lipofectamine 2000 (33.14 ng luciferase/mg protein). pDNA-derived gene transfer to superficial burn wounds showed the highest expression among burn groups (0.77 ng luciferase/mg protein). However, lipoplex-derived gene transfer to superficial burns and unburned skin failed to show higher expression. CONCLUSIONS: Lipofectamine 2000 and DOTAP/Chol lipoplex showed significantly enhanced gene transfer, whereas no transfection was detectable for naked DNA in vitro. In contrast to the in vitro study, naked DNA was the only agent with which gene delivery was successful in experimental burn wounds. These findings highlight the limited predictability of in vitro analysis for gene delivery as a therapeutic approach.     

Study on the Multidrug Resistance 1 Gene Transfection Efficiency Using Adenovirus Vector Enhanced by Ultrasonic Microbubbles In Vitro

As gene delivery reagents, microbubbles have been successfully used in combination with ultrasound. Shock wave exposure has been shown to transfect cells with naked DNA in vitro, but it has not been tested whether the addition of microbubbles would enhance DNA uptake with adenovirus vector. Therefore, the aim of this study was to study the efficacy and safety of multidrug resistance 1 (MDR1) gene transfer into the bone marrow mononuclear cells of rabbits using adenovirus vector enhanced by ultrasound with microbubbles in vitro. The transfection rate of the MDR1 gene was significantly increased by ultrasound microbubbles with adenovirus. After ultrasonic irradiation, there were transient holes in the cell membrane, which disappeared after irradiation by ultrasound for 24 h. The temporary swelling of the organelles was reversible. Our in vitro findings conclusively demonstrate that the exogenous MDR1 gene transfer into the mononuclear cells of rabbits with adenovirus vector was enhanced by the ultrasonic microbubbles and this transfection technique is safe.     

Protein transduction domain delivery of therapeutic macromolecules

Owing to their unprecedented selectivity, specific activity and potential for 1000+ fold amplification of signal, macromolecules, such as peptides, catalytic protein domains, complete proteins, and oligonucleotides, offer great potential as therapeutic molecules. However, therapeutic use of macromolecules is limited by their poor penetration in tissues and their inability to cross the cellular membrane. The discovery of small cationic peptides that cross the membrane, called Protein Transduction Domains (PTDs) or Cell Penetrating Peptides (CPPs), in the late 1980s opened the door to cellular delivery of large, bioactive molecules. Now, PTDs are widely used as research tools, and impressively, multiple clinical trials are testing PTD-mediated delivery of macromolecular drug conjugates in patients with a variety of diseases.