1,4-Butanediol diglycidyl ether (BDE)-crosslinked PEI-g-imidazole nanoparticles as nucleic acid-carriers in vitro and in vivo

Previously reported 1,4-butanediol diglycidyl ether (BDE) crosslinked PEI (branched polyethylenimine, 25 k) nanoparticles (A. Swami, R. Kurupati, A. Pathak, Y. Singh, P. Kumar and K. C. Gupta, A unique and highly efficient non-viral DNA/siRNA delivery system based on PEI-bisepoxide nanoparticles, Biochem. Biophys. Res. Commun., 2007, 362, 835-841) (PN NPs) were reacted with varying proportions of a novel linker, 2-(N-1-tritylimidazol-4-yl)-N-(6-glycidyloxyhexyl)-acetamide (IGA linker, 3), to yield PN-g-imidazolyl nanoparticles (PNIm) with improved transfection efficiency. Here, the IGA linker (3) reacted through an epoxy ring to partially convert the residual 1° and 2° amines present in PN NPs to 2° and 3°, respectively, without altering the total number of amines and additionally incorporating the delocalized positive charge of the imidazolyl moiety. The resulting particles were characterized for their size, zeta potential and DNA complexing ability. PNIm/DNA nanoplexes, in the size range of 120-400 nm, were evaluated for transfection efficiency in HeLa, HEK293 and CHO cell lines, which was found to be ～11, ～2-3 and ～2-17 folds higher than PEI, PN-2 (the best working sample of the PN series) (A. Swami, R. Kurupati, A. Pathak, Y. Singh, P. Kumar and K. C. Gupta, A unique and highly efficient non-viral DNA/siRNA delivery system based on PEI-bisepoxide nanoparticles, Biochem. Biophys. Res. Commun., 2007, 362, 835-841) and commercial transfection reagents tested in this study, respectively. Also, flow cytometric analysis showed ～78% (ca.～43% in PN-2) cells transfected with the PNIm 10(6)/DNA complex (the best working sample of the PNIm series) in HEK293 cells. Transfection of GFP specific siRNA in HEK293 cells suppressed the gene expression by ～90% (ca.～70% in PN-2). All the cell lines treated with PNIm/DNA nanoplexes showed >90% viability. In vivo gene expression of luciferase enzyme in Balb/c mice showed highest expression in spleen after seven days.     

Alginate modified nanostructured calcium carbonate with enhanced delivery efficiency for gene and drug delivery

To improve the performance of nanostructured calcium carbonate in gene delivery, a hydrophilic polysaccharide, alginate, was added to calcium carbonate co-precipitation systems to form alginate/CaCO(3)/DNA nanoparticles. The size and ζ-potential of the nanoparticles were measured by a zetasizer. Due to the existence of alginate chains which retarded the growth of calcium carbonate based co-precipitates, the alginate/CaCO(3)/DNA nanoparticles exhibited a decreased size and enhanced stability in the aqueous solution. To evaluate the gene and drug co-delivery ability, doxorubicin hydrochloride (DOX), a water-soluble anticancer drug, was loaded in the nanoparticles to form alginate/CaCO(3)/DNA/DOX nanoparticles. The in vitro gene transfections mediated by different nanoparticles in 293 T cells and HeLa cells were carried out, using pGL3-Luc as a reporter plasmid. With an appropriate amount of alginate, the gene transfection efficiency of alginate modified nanoparticles could be significantly enhanced as compared with the nanoparticles without alginate modification for the gene delivery systems, as well as the gene and drug co-delivery systems. The study on in vitro cell inhibition effects showed that the cell viability decreased with increasing DOX amount loaded in alginate/CaCO(3)/DNA/DOX nanoparticles. The alginate modification is a useful strategy to improve the calcium carbonate co-precipitation technique for the preparation of gene and drug delivery systems, and the nanoparticles prepared in this study have promising applications in gene and drug delivery.     

The methyl donor S-Adenosylmethionine inhibits active demethylation of DNA: a candidate novel mechanism for the pharmacological effects of S-Adenosylmethionine

S-Adenosylmethionine (AdoMet) is the methyl donor of numerous methylation reactions. The current model is that an increased concentration of AdoMet stimulates DNA methyltransferase reactions, triggering hypermethylation and protecting the genome against global hypomethylation, a hallmark of cancer. Using an assay of active demethylation in HEK 293 cells, we show that AdoMet inhibits active demethylation and expression of an ectopically methylated CMV-GFP (green fluorescent protein) plasmid in a dose-dependent manner. The inhibition of GFP expression is specific to methylated GFP; AdoMet does not inhibit an identical but unmethylated CMV-GFP plasmid. S-Adenosylhomocysteine (AdoHcy), the product of methyltransferase reactions utilizing AdoMet does not inhibit demethylation or expression of CMV-GFP. In vitro, AdoMet but not AdoHcy inhibits methylated DNA-binding protein 2/DNA demethylase as well as endogenous demethylase activity extracted from HEK 293, suggesting that AdoMet directly inhibits demethylase activity, and that the methyl residue on AdoMet is required for its interaction with demethylase. Taken together, our data support an alternative mechanism of action for AdoMet as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA.     

Transfection of partially purified plasmid DNA for high level transient protein expression in HEK293-EBNA cells

One of the major constraints to performing large-scale transfections of cultured mammalian cells for the transient expression of recombinant proteins is the production of large quantities of purified plasmid DNA. In this report partially purified plasmid DNA was prepared by a method that combines alkaline lysis of E. coli with standard precipitation techniques. The efficiency of calcium phosphate-DNA co-precipitate formation with crude DNA was similar to that observed for pure DNA, but precipitate formed with crude DNA also contained RNA. The transfection of adherent and suspension-adapted HEK293-EBNA cells with partially purified pEGFPN1 resulted in levels of transient GFP expression equivalent to those achieved with pure DNA. In addition, the co-transfection of 1-200 ml cultures of suspension-adapted HEK293-EBNA cells with two different plasmids encoding the heavy and light chain genes of anti-human RhD IgG1, respectively, yielded similar IgG titers with pure and partially purified plasmid DNA. Finally, it was observed that suspension-adapted cells were more tolerant to the presence of RNA in the plasmid preparations than were adherent cells. These findings are relevant to the field of DNA transfection, including applications ranging from high-throughput screening to large-scale transient protein expression.     

Production of lentiviral vectors in suspension cells using low proportion of supercoiled circular plasmid DNA

The supercoiled circular (SC) topology form of plasmid DNA has been regarded to be advantageous over open circular or linearized analogue in transfection and expression efficiency, and therefore are largely demanded in the biopharmaceutical manufacturing. However, production of high-purity SC plasmid DNA would result in high manufacturing cost. The effect of SC proportion in plasmid DNA on the quality of packaged lentiviral vectors has never been reported. In this study, we established an efficient system for production of high-titer lentiviral vectors using suspension HEK293SF cells in serum-free media, and the lentiviral titer was not associated with the proportion of SC plasmid DNA. Plasmids DNA with different proportion of SC, open-circular, and linearized forms were prepared using the thermal denaturation method, and were transfected to adherent HEK293T or suspension HEK293SF cells for packaging of lentiviral vectors. The titer of lentiviral vectors from HEK293T cells, but not from HEK293SF cells, was significantly impaired when the proportion of SC plasmid DNA decreased from 60–80% to 30–40%. Further decrease of SC plasmid proportion to 3% led to a dramatic reduction of lentiviral titer no matter the packaging cell line was. However, lentiviral vectors from HEK293SF cells still showed a high titer even when the proportion of SC plasmid DNA was 3%. This study demonstrated that extremely high proportion of SC plasmid DNA was not required for packaging of high-titer lentiviral vector in HEK293SF cells, at least under our manufacturing process.     

Quaternary complexes composed of plasmid DNA/protamine/fish sperm DNA/stearic acid grafted chitosan oligosaccharide micelles for gene delivery

Quaternary complexes with condensed core of plasmid DNA, protamine, fish sperm DNA and shell of stearic acid grafted chitosan oligosaccharide (CSO-SA), were prepared. The CSO-SA could self-assemble to form nano-sized micelles in aqueous solution and demonstrated excellent internalization ability of tumor cells. Dynamic light scattering (DLS) measurement and transmission electrostatic microscope (TEM) images showed that quaternary complexes had spherical shape with about 25 nm number average diameter, and the size of quaternary complexes was smaller than that of CSO-SA micelles and CSO-SA micelles/plasmid DNA binary complexes. The transfection efficiencies of quaternary complexes on HEK293 and MCF-7 cells increased with incubation time, and were significantly higher than that of CSO-SA micelles/plasmid DNA binary complexes. The optimal transfection efficiency of quaternary complexes on HEK293 and MCF-7 cells measured by flow cytometer after 96 h was 23.82% and 41.43%, respectively. Whereas, the transfection efficiency of Lipofectamine? 2000 on HEK293 and MCF-7 cells after 96 h was 32.45% and 33.23%, respectively. The data of luciferease activity measurement showed that the optimal ratio of plasmid DNA:fish sperm DNA:protamine:CSO-SA was 1:1:5:5. The results indicated that the present quaternary complexes were potential non-viral gene delivery system.     

Tuftsin-modified alginate nanoparticles as a noncondensing macrophage-targeted DNA delivery system

The main objective of this study was to evaluate macrophage-targeted alginate nanoparticles as a noncondensing gene delivery system for potential anti-inflammatory therapy. An external gelation method was employed to form plasmid DNA-encapsulated alginate nanoparticles. The nanoparticle surface was modified with a peptide sequence containing tuftsin (TKPR), and transfection efficiency was determined in J774A.1 macrophages. The effect of transfected mIL-10 in blocking expression of tumor necrosis factor-alpha (TNF-α) was evaluated in lipopolysaccharide (LPS)-stimulated cells. Scrambled peptide- and tuftsin-modified cross-linked alginate nanoparticles efficiently encapsulated plasmid DNA and protected against DNase I degradation. The transgene expression efficiencies, measured using GFP and mIL-10 expressing plasmid DNA, were highest with tuftsin-modified nanoparticles. Levels of TNF-α were significantly lower (p < 0.0001) in LPS-stimulated cells that were transfected with mIL-10 using alginate nanoparticles. The results of the study show that noncondensing alginate nanoparticles can efficiently deliver plasmid DNA, leading to sustained in vitro gene expression in macrophages.     

Fabrication and Optimization of Linear PEI-Modified Crystal Nanocellulose as an Efficient Non-Viral Vector for In-Vitro Gene Delivery

Background:One of the major challenges in gene therapy is producing gene carriers that possess high transfection efficiency and low cytotoxicity (1). To achieve this purpose, crystal nanocellulose (CNC) -based nanoparticles grafted with polyethylenimine (PEI) have been developed as an alternative to traditional viral vectors to eliminate potential toxicity and immunogenicity.Methods:In this study, CNC-PEI10kDa (CNCP) nanoparticles were synthetized and their transfection efficiency was evaluated and compared with linear cationic PEI10kDa (PEI) polymer in HEK293T (HEK) cells. Synthetized nanoparticles were characterized with AFM, FTIR, DLS, and gel retardation assays. In-vitro gene delivery efficiency by nano-complexes and their effects on cell viability were determined with fluorescent microscopy and flow cytometry.Results:Prepared CNC was oxidized with sodium periodate and its surface cationized with linear PEI. The new CNCP nano-complex showed different transfection efficiencies at different nanoparticle/plasmid ratios, which were greater than those of PEI polymer. CNPC and Lipofectamine were similar in their transfection efficiencies and effect on cell viability after transfection.Conclusion:CNCP nanoparticles are appropriate candidates for gene delivery. This result highlights CNC as an attractive biomaterial and demonstrates how its different cationized forms may be applied in designing gene delivery systems.Key Words: Crystal Nanocellulose, Gene transfection, Nanoparticle, Nano-complex     

Characterization of multilayered nanoparticles encapsulated in yeast cell wall particles for DNA delivery

Nonviral gene delivery technologies have been developed using layer-by-layer self-assembly of nanomaterials held together by electrostatic interactions in order to provide nanoparticulate materials that protect and deliver DNA to cells. Here we report a new DNA delivery technology based on the in situ layer-by-layer synthesis of DNA nanoparticles caged within hollow yeast cell wall particles (YCWP). YCWP provide protection and facilitate oral and systemic receptor-targeted delivery of DNA payloads to phagocytic cells. The nanoparticles inside YCWP consist of a core of tRNA/polyethylenimine (PEI) followed by a DNA layer that is finally coated with a protective outer layer of PEI. Using fluorescein and rhodamine labeling of tRNA, PEI, and DNA, the layer-by-layer formation of the nanoparticles was visualized by fluorescent microscopy and quantitated by fluorescence spectroscopy and flow cytometry. Optimal conditions (tRNA:YCWP, PEI:YCWP ratios and DNA load levels) to synthesize YCWP encapsulated nanoparticles were determined from these results. The high in vitro transfection efficiency of this encapsulated DNA delivery technology was demonstrated by the transfection of NIH3T3-D1 cells with YCWP-tRNA/PEI/gWizGFP/PEI formulations containing low amounts of the plasmid gWizGFP per particle to maximally express green fluorescent protein (GFP).     

Synthesis and characterization of folate-PEG-grafted-hyperbranched-PEI for tumor-targeted gene delivery

A great challenge for gene therapy is to develop a high efficient gene delivery system with low toxicity. Nonviral vectors are still attractive although the current agents displayed some disadvantages (i.e., low transfection efficiency, high toxicity). To overcome the high toxicity of poly(ethylene imine) (PEI) and low transfection efficiency of PEGylated PEI (PEG-PEI), we linked a cell specific target molecule folate (FA) on poly(ethylene glycol) (PEG) and then grafted the FA-PEG onto hyperbranched PEI 25 kDa. The FA-PEG- grafted-hyperbranched-PEI (FA-PEG-PEI) effectively condensed plasmid DNA (pDNA) into nanoparticles with positive surface charge under a suitable N/P ratio. Tested in deferent cell lines (i.e., HEK 293T, glioma C6 and hepatoma HepG2 cells), no significant cytotoxicity of FA-PEG-PEI was added to PEG-PEI. More importantly, significant transfection efficiency was exhibited in FA-targeted cells. Reporter assay showed that FA-PEG-PEI/pDNA complexes had significantly higher transgene activity than that of PEI/pDNA in folate-receptor (FR) positive (HEK 293T and C6) cells but not FR-negative (HepG2) cells. These results indicated that FA-PEG-PEI might be a promising candidate for gene delivery with the characteristics of good biocompatibility, potential biodegradability, and relatively high gene transfection efficiency.     

Synthesis of novel biodegradable cationic polymer: N,N-diethylethylenediamine polyurethane as a gene carrier

A new cationic polymer, N,N-diethylethylenediamine-polyurethane (DEDA-PU), bearing tertiary amines in the backbone and side chains, was synthesized and used as a nonviral vector for gene delivery. The DEDA-PU readily self-assembled with the plasmid DNA (pCMV-betagal) in water and buffer at physiological pH, as determined by agarose gel retardation, dynamic light scattering, zeta potential, atomic force microscopy (AFM), and restriction endonuclease protection assays. The results revealed that DEDA-PU was able to bind with plasmid DNA, yielding positively charged complexes with a size around 100 nm at a DEDA-PU/DNA ratio of 50/1 (w/w). The DEDA-PU/DNA complexes were able to transfect HEK 293 cells in vitro with an efficiency comparable to a well-known gene carrier [poly(2-dimethylaminoethyl methacrylate), PDMAEMA]. The cytotoxicity of DEDA-PU was substantially lower than PDMAEMA. The degradation studies indicated that DEDA-PU degrades hydrolytically in 20 mM HEPES buffer at pH 7.4 with a half-life of approximately 60 h. This study shows that DEDA-PU holds promise as biodegradable polycations for gene delivery and is interesting candidate for further study.     

Expression and analysis of green fluorescent proteins in human embryonic kidney cells by capillary electrophoresis

The green fluorescent protein (GFP) has attracted much interest as a reporter for gene expression. In this paper, application of capillary electrophoresis with laser-induced fluorescent (CE-LIF) for quantitation of green fluorescence protein in cellular extracts and single cells is investigated. The S65T mutant form of GFP protein was successfully expressed in human embryonic kidney (HEK293) cells, and its production was confirmed by fluorescence microscopy and CE-LIF. The mass limit of detection for the mutant S65T was 5.3 x 10(-20) mol, which was better than that for the wild-type GFP by a factor of six. Detection of a small amount of GFP is difficult by conventional techniques such as fluorescent microscopy due to interference from cell autofluorescence at low GFP concentrations. The HEK293 cells were transfected with the GFP plasmid that produced S65T-GFP. Transient production of S65T protein was detected 2 h after the transfection and reached a maximum after 48 h. The protein concentration began to decrease significantly after 96 h. Single cell analysis of HEK293 cells after transfection with GFP plasmid indicate a nonuniform production of S65T-GFP protein among cells.     

超正电荷绿荧光蛋白+36GFP作为核酸载体的细胞穿透性分析

旨在通过原核表达纯化超正电荷绿色荧光蛋白+36GFP,研究其与核酸的结合作用及作为核酸载体的细胞转导功能。将pET+36GFP-HA2质粒转化到大肠杆菌BL21(DE3)菌株中,然后表达纯化+36GFP蛋白。将得到的目的蛋白在特定浓度下分别转导293细胞、HepG2细胞、A549细胞和B16细胞,流式细胞仪检测+36GFP的转导效率;+36GFP蛋白(100 nmol/L)转导A549细胞,激光共聚焦显微镜观察结果;将+36GFP蛋白与质粒DNA按不同比例孵育,凝胶阻滞实验检测+36GFP与DNA的结合能力;激光共聚焦显微镜和流式细胞仪检测+36GFP蛋白携带质粒DNA转导细胞后报告基因的表达。结果显示,+36GFP蛋白具有较高的细胞转导效率,且随浓度升高转导效率增加,呈浓度依赖性。凝胶阻滞实验显示,+36GFP能够与质粒DNA结合,阻滞DNA在凝胶中迁移,且呈现一定的浓度依赖性。+36GFP包裹质粒转导细胞后,可高效携带质粒DNA转导进入细胞,使质粒报告基因得到表达。本研究成功表达纯化了+36GFP蛋白,证实该蛋白具有较高的细胞转导效率,可将外源核酸携带入细胞使外源基因得到表达。     

转录因子Twist的siRNA筛选、腺病毒构建及功能检测

目的 筛选特异性沉默人的Twist基因的siRNA序列,构建siTwist腺病毒并在MG63及143B骨肉瘤细胞中进行功能鉴定.方法 体外退火获得4组siTwist双链DNA序列,克隆至含有Twist基因的pSOS-Twist质粒中获得pSOS-siTwist质粒,脂质体转染HEK293细胞,GFP检测筛选有功能的siTwist片段,将筛选出的siTwist序列构建腺病毒,感染143B骨肉瘤细胞.通过RT-PCR、Western 印迹检测Twist的表达.siTwist与Twist腺病毒共感染MG63骨肉瘤细胞,细胞计数及细胞侵袭实验检测siTwist对Twist的抑制作用.结果 在HEK293细胞中,4组siTwist中有2组GFP的表达明显降低,且siTwist腺病毒能抑制143B骨肉瘤细胞中内源性的Twist表达,Twist腺病毒能促进MG63骨肉瘤细胞的增殖和转移,而两组siTwist与Twist共感染组MG63细胞的增殖及迁徙率均明显低于Twist组（P〈0.05）.结论 筛选出两对特异性沉默Twist基因的siRNA片段,并成功构建腺病毒,转染细胞后能有效抑制内源性和外源性的Twist表达,为研究Twist在骨肉瘤细胞增殖和转移中的作用及具体机制提供了有效的分子工具.     

Exploring N-imidazolyl-O-carboxymethyl chitosan for high performance gene delivery

Chitosan shows good biocompatibility and biodegradability, but the poor water solubility and low transfection efficiency hinder its applications as a gene delivery vector. We here report the detailed synthesis and characterization of a novel ampholytical chitosan derivative, N-imidazolyl-O-carboxymethyl chitosan (IOCMCS), used for high performance gene delivery. After chemical modification, the solubility of the resulting polymer is enhanced, and the polymer is soluble in a wide pH range (4-10). Gel electrophoresis study reveals the strong binding ability between plasmid DNA and the IOCMCS. Moreover, the IOCMCS does not induce remarkable cytotoxicity against human embryonic kidney (HEK293T) cells. The cell transfection results with HEK293T cells using the IOCMCS as gene delivery vector demonstrate the high transfection efficiency, which is dependent on the degree of imidazolyl substitution. Therefore, the IOCMCS is a promising candidate as the DNA delivery vector in gene therapy due to its high solubility, high gene binding capability, low cytotoxicity, and high gene transfection efficiency.     

Characterization of (aminoethyl)chitin/DNA nanoparticle for gene delivery

Nonviral gene delivery systems have been increasingly proposed as a safer alternative to viral vehicles. In the present study, we synthesized water-soluble chitin by aminoalkylating onto chitin at the C-6 position, and its transfection efficiency was investigated. Aminoethyl-chitin (AEC) was complexed with DNA, and AEC/DNA nanoparticles were characterized. AEC/DNA nanoparticles showed good DNA binding ability, high protection of DNA from nuclease and serum, and low cytotoxicity. Mean particle size decreased from 367 to 290 nm and zeta potential increased from -4.58 to 22.87 mV when the AEC/DNA charge ratio (N/P) increased from 1.15 to 18.5. The transfection efficiency of AEC/DNA nanoparticles was investigated in a human embryonic kidney cell line (HEK293), and the results showed that AEC/DNA nanoparticles were much enhanced compare with naked DNA.     

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.     

反转录病毒载体介导的猪APOBEC3F在猪源细胞PK15中的表达

目的:利用反转录病毒载体构建猪载脂蛋白B mRNA编辑酶催化多肽样蛋白(APOBEC)3F重组质粒,并实现其在猪肾细胞PK15中的表达。方法:用RT-PCR方法扩增五指山猪来源的外周血淋巴细胞APOBEC3F基因,将其定点插入反转录病毒载体pMSCV neo中,同时于插入位点两侧分别添加FLAG和GFP标签,构建重组质粒pMSCV-FLAG-A3F-GFP,并进行酶切、测序鉴定;将鉴定正确的重组质粒与pVSV-G、pGag-Pol共转染包装细胞HEK293T,分别于转染后48~72 h收集细胞的培养上清以获得假型病毒粒子;用该假型病毒感染猪源细胞PK15,通过PCR、Western印迹检测目的基因的整合及表达。结果:PCR扩增到1254 bp的猪APOBEC3F基因,重组质粒pMSCV-FLAG-A3F-GFP经酶切、测序,结果无误;3质粒共转染HEK293T细胞包装出的假型病毒感染PK15细胞后观察到GFP表达;从感染假型病毒的PK15细胞基因组中扩增到1254 bp的猪APOBEC3F基因,Western印迹检测到78.1×103的猪APOBEC3F蛋白的表达。结论:实现了反转录病毒载体介导的猪APOBEC3F在猪源细胞PK15中的整合与表达,为深入研究该分子对猪内源性反转录病毒(PERV)的抑制作用奠定了基础。