Chitosan-Based Vector/DNA Complexes for Gene Delivery: Biophysical Characteristics and Transfection Ability

Purpose. Chitosan, a natural cationic polysaccharide, is a candidate non-viral vector for gene delivery. With the aim of developing this system, various biophysical characteristics of chitosan-condensed DNA complexes were measured, and transfections were performed. Methods. Transmission electronic microscopy (TEM) visualizations, sedimentation experiments, dynamic light scattering (DLS), and zeta potential measurements were realized. Transfections were made by using the luciferase reporter gene. Results. In defined conditions, plasmid DNA formulated with chitosan produced homogenous populations of complexes which were stable and had a diameter of approximately 50–100 nm. Discrete particles of nicely condensed DNA had a donut, rod, or even pretzel shape. Chitosan/DNA complexes efficiently transfected HeLa cells, independently of the presence of 10% serum, and did not require an added endosomolytic agent. In addition, gene expression gradually increased over time, from 24 to 96 hours, whereas in the same conditions the efficacy of polyethylenimine-mediated transfection dropped by two orders of magnitude. At 96 hours, chitosan was found to be 10 times more efficient than PEI. However, chitosan-mediated transfection depended on the cell type. This dependency is discussed here. Conclusions. Chitosan presents some characteristics favorable for gene delivery, such as the ability to condense DNA and form small discrete particles in defined conditions.     

Targeted Delivery of Complexes of Biotin–PEG–Polyethylenimine and NF-κB Decoys to Brain-derived Endothelial Cells <Emphasis Type="BoldItalic">in Vitro</Emphasis>

Purpose To evaluate the effect of re-directing the uptake mechanism of polyplexes containing oligodeoxynucleotide (ODN) decoys to nuclear factor kappa B (NF-κB) from absorptive-mediated to receptor-mediated endocytosis. Materials and Methods Complexes of ODNs and a co-polymer of biotin–polyethylenglycol and polyethylenimine (BPP) were targeted to brain-derived endothelial cells with a conjugate of antibody 8D3 and streptavidin (8D3SA). Size and stability of ODN/BPP complexes was measured by dynamic light scattering. Cellular uptake was studied by confocal microscopy. Cell viability and pharmacological effects were investigated on murine bEnd5 cells stimulated with tumor necrosis factor. Results ODN/BPP complexes showed sizes of 116 ± 2.3 nm, which increased by 40 nm when coupled to 8D3SA, and were stable in physiological fluids. Targeted complexes were internalized intact into endosomal compartments. Treatment conditions, which yielded significant inhibitory effects on mRNA expression of VCAM-1, ICAM-1, IκBα and iNOS by bEnd5 cells, did not affect viability. At 0.5 μM, decoy ODN significantly inhibited monocyte adhesion to bEnd5 monolayers when delivered as 8D3SA-targeted complex, while higher concentrations of untargeted complex were ineffective. Conclusions The complex of NF-κB decoys and BPP, which can be targeted to transferrin receptors, is a promising drug candidate for neuroinflammatory diseases affecting the blood–brain barrier.     

Synthesis and evaluation of a water‐soluble polymer to reduce Ac‐225 daughter migration

The actinium decay chain has been promoted as an in vivo alpha generator for therapy, but migration of daughters from the primary conjugate has lead to increased toxicity away from the target organ. To reduce daughter migration, polyethylenimine (PEI) was used with a primary chelator and secondary chelators. The primary chelator, DOTA, was used to coordinate ²²⁵Actinium and secondary chelators‐acetate and DTPA, were added to the polymer for coordination of daughters formed by decay. The ²²⁵Actinium polymer derivatives containing secondary chelators were found to retain radioactive daughters better than the ²²⁵Actinium bond to the primary alone. The retention of ²¹³Bismuth and ²⁰⁹Thallium had the following order from highest retained to lowest DOTA‐PEI‐DTPA≈DOTA‐PEI‐CH₂OO‐ > DOTA‐PEI. The data suggests this polymer approach could be used to reduce daughter migration and has potential for development of actinium labeled radiopharmaceuticals. Copyright © 2007 John Wiley & Sons, Ltd.     

Methoxy poly(ethylene glycol) – Low molecular weight linear polyethylenimine-derived copolymers enable polyplex shielding

Targeted gene delivery relies on the development of materials that allow for the formation of small neutrally charged particles of sufficient colloidal stability preventing non-specific interactions with cells. In order to identify a copolymer composition that combines adequate plasmid DNA (pDNA) compaction with an efficient charge-shielding effect, we synthesized a series of copolymers by covalent linkage of activated 5 or 20 kDa linear methoxy poly(ethylene glycol) (mPEG) or 10 kDa two-arm-mPEG to non-toxic low molecular weight (2.6 and 4.6 kDa) linear polyethylenimine (lPEI) at different molar ratios (mPEG–lPEI copolymers). All of the copolymers condensed pEGFP-N1 pDNA to form nanoparticles with hydrodynamic diameters between 150 and 420 nm – sizes that were maintained for the entire duration of measurement. PEGylated complexes exhibited a reduced particle stability in comparison to the unmodified lPEI–pDNA polyplexes, determined by gel retardation assays and DNase I experiments. Copolymer–pDNA complexes exhibited a zeta potential between −4 and 6 mV, strongly depending on the dispersion medium applied (0.15 M NaCl or 5% glucose supplemented with serum-free cell culture medium). The transfection efficacy, determined in CHO-K1 (between 0.28 ± 0.08% and 1.92 ± 0.46%) and HeLa (between 1.02 ± 0.19% and 3.53 ± 0.30%) cells, was significantly reduced compared to lPEI–pDNA particles (between 3.2 ± 1.3% and 38.8 ± 5.5%). The architecture of the copolymer, the molecular weight of the lPEI residue, and the supplementation of endosomolytic agents (saccharose, chloroquine) all failed to impact the efficacy of gene transfer. Uptake studies, based on Confocal Laser Scanning Microscopy (CLSM) imaging and flow cytometry analysis, suggest that the use of mPEG5/3–lPEI2.6, mPEG10/2–lPEI2.6, and mPEG20–lPEI4.6 lowers unspecific internalization of the corresponding transfection complexes. This provides an ideal basis for the development of transfection vehicles for targeted gene transfer.     

Stabilized Nonviral Formulations for the Delivery of MCP-1 Gene into Cells of the Vasculoendothelial System

PURPOSE: The purpose of this study was to develop a stabilized non-viral gene transfer system for the efficient delivery and expression of monocyte chemoattractant protein 1 (MCP-1) gene in cells of the vasculoendothelial system. METHODS: Plasmid DNA was condensed with polyethylenimine (PEI), conjugates of PEI with polyethylene glycol (PEG), and PEI conjugates with the membrane-active peptide melittin. Surface charge and particle size of the resulting gene transfer particles were analyzed by laser light scattering. Reporter gene studies and toxicity assays were conducted on smooth muscle cells and endothelial cells of human, porcine, or rat origin. RESULTS: Nonviral gene carriers containing PEI and PEG were developed that could be produced in batches of several milligrams and conveniently stored as frozen samples. Incorporation of PEG into the transfection complex significantly reduced cellular toxicity. The cryoconserved gene transfer particles mediated high expression of luciferase, enhanced green fluorescent protein (EGFP), or secreted alkaline phosphatase reporter genes. Highest reporter gene expression was achieved with PEI polyplexes containing PEG and melittin. The gene for MCP-1 was efficiently delivered into target cells and resulted in expression of up to 125 ng/ml secreted bioactive MCP-1 protein per 50,000 cells. CONCLUSIONS: Gene carriers based on PEI and PEG display reduced toxicity, can be stored in frozen form without loss of biological activity, and can efficiently transfect cells of the vasculoendothelial system. Such gene carriers hold a potential for use in arterial gene transfer and local secretion of MCP-1 as trigger of therapeutic arteriogenesis in arterial occlusion diseases.     

DNA/polyethylenimine transfection particles: Influence of ligands, polymer size, and PEGylation on internalization and gene expression

Receptor-binding ligands have been incorporated into DNA/polyethylenimine (PEI) complexes to enhance cell binding and cellular internalization. This study characterizes receptor-mediated uptake of DNA/PEI complexes on a cellular basis. A novel assay based on flow cytometry was applied, discriminating between total cell-associated and extracellularly bound DNA complexes. Receptor-mediated uptake of ligand-containing DNA/PEI (molecular weight, 800 kd) complexes was found to occur quickly (within 1 hour), whereas unspecific uptake through adsorptive endocytosis is less efficient or requires extended periods to reach the same degree of internalization. Rapid, receptor-mediated internalization requires a small complex size; however, large, aggregated complexes show higher gene expression. Using PEI 25 kd conjugated to large proteins such as transferrin or antibodies, improper condensation with DNA leads to suboptimal uptake and gene expression, whereas partial replacement of ligand-PEI with unconjugated PEI increases both uptake and transfection. In contrast, the 8 kd protein epidermal growth factor conjugated to PEI 25 kd properly condenses DNA and mediates specific uptake into human adenocarcinoma (KB) cells. Modification of the complex surface with appropriate amounts of poly(ethylene glycol) (PEG) does not block ligand-mediated internalization. A higher degree of PEGylation reduces the internalization of transferrin or antibody-containing complexes to a level similar to that of ligand-free complexes. In contrast, epidermal growth factor-mediated uptake is less effected by excessive PEGylation.     

藻酸盐/PEI/DNA复合载体作为一种新型基因递送系统

目的克服多聚乙烯亚胺(PEI，polyethlenimine)/DNA载体对细胞的毒性以及在含血清培养基里对癌细胞基因的转移率低的问题。方法利用具有水溶性、可生物降解的、并带有负电的藻酸盐(alginate)对PEI/DNA载体进行包衣，制备出复合载体，并在体外含50%血清培养基里，与PEI/DNA载体比较对C3癌细胞转染率。结果 在含50%血清的培养基里，藻酸盐包衣制备的复合体载体［alginate:DNA，0.15 (w/w);PEI:DNA，N:P=10］与PEI/DNA载体相比，对C3癌细胞基因转染率高出10～30倍，而且其表面正电荷数比PEI/DNA载体减少了一半，颗粒较小，并降低对细胞毒性和红血球集聚反应。结论作为新型的藻酸盐包衣制备的复合载体能提高在体外含高浓度血清培养基里对C3癌细胞的转染率，并能减少其对细胞毒性。     

多聚乙烯基亚胺介导的pOSP1-HSVtk基因对卵巢癌的抗瘤研究

目的: 研究多聚乙烯基亚胺(PEI)介导的卵巢特异性启动子调控下的自杀基因对卵巢癌的抗肿瘤作用.方法:(1)将卵巢特异性启动子调控下的真核表达质粒pOSP1-HSVtk利用PEI导入人卵巢癌细胞SKOV3,人肺癌细胞NCI-H460和人肝癌细胞HepG2,MTT法测定GCV对三种肿瘤细胞的杀伤作用;(2)建立卵巢癌移植瘤模型,瘤周注射PEI/pOSP1-HSVtk复合物,通过HPLC监测体内GCV浓度的变化来评价TK基因在肿瘤组织中的表达效率;同时记录裸鼠的体重、瘤体大小及瘤重,计算体积和重量抑瘤率,并进行肿瘤组织的病理学和TUNEL检测.结果: (1)GCV仅对SKOV3细胞具有杀伤作用;(2)与对照组相比,GCV的浓度在转染pOSP1-HSVtk的肿瘤组织局部显著降低(0.05>P>0.01);治疗组的肿瘤体积和瘤重显著减小(P<0.01),体积抑瘤率与重量抑瘤率分别为63.66%和58.98%.组织学示治疗组肿瘤细胞有出血及坏死,TUNEL证实了细胞的凋亡.结论: 多聚乙烯基亚胺介导的卵巢特异性启动子调控下的自杀基因对卵巢癌有靶向杀伤作用.     

Covalent binding of the anticancer drug ellipticine to DNA in V79 cells transfected with human cytochrome P450 enzymes

Ellipticine is a potent antineoplastic agent whose mechanism of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Recently, we found that ellipticine also forms covalent DNA adducts and that the formation of the major adduct is dependent on the activation of ellipticine by cytochrome P450 (CYP). We examined a panel of genetically engineered V79 cell lines including the parental line V79MZ and recombinant cells expressing the human CYP enzymes CYP1A1, CYP1A2 or CYP3A4 for their ability to activate ellipticine. The extent of activation was determined by analysing DNA adducts by 32P-postlabelling. Ellipticine was found to be toxic to all V79 cell lines with IC(50) values ranging from 0.25 to 0.40 microM. The nuclease P1 version of the 32P-postlabelling assay yielded a similar pattern of ellipticine-DNA adducts with two major adducts in all cells, the formation of only one of which was dependent on CYP activity. This pattern is identical to that detected in DNA reacted with ellipticine and the reconstituted CYP enzyme system in vitro as confirmed by HPLC of the isolated adducts. Total adduct levels ranged from 2 to 337 adducts per 10(8) nucleotides, in the parental line and in V79 expressing CYP3A4, respectively. As in vitro, human CYP1A2 and CYP1A1 were less active. The results presented here are the first report showing the formation of CYP-mediated covalent DNA adducts by ellipticine in cells in culture, and confirm the formation of covalent DNA adducts as a new mechanism of ellipticine action.     

A Novel Non-Viral Vector for DNA Delivery Based on Low Molecular Weight,Branched Polyethylenimine: Effect of Molecular Weight on Transfection Efficiency and Cytotoxicity

Purpose. Low molecular weight branched polyethylenimine (LMW-PEI) was synthesized and studied as a DNA carrier for gene delivery with regard to physico-chemical properties, cytotoxicity, and transfection efficiency. Methods. The architecture of LMW-PEI, synthesized by acid catalyzed ring-opening polymerization of aziridine was characterized by size exclusion chromatography in combination with laser light scattering and ¹³C-NMR-spectroscopy. In vitro cytotoxic effects were quantified by LDH and MTT assay and visualized by transmission electron microscopy. The potential for transgene expression was monitored in ECV304 cells using luciferase driven by a SV40 promoter as reporter gene system. Results. LMW-PEI (Mw 11900 D) with a low degree of branching was synthesized as a DNA carrier for gene delivery. In contrast to high molecular weight polyethylenimines (HMW-PEI; Mw l616OOO D), the polymer described here showed a different degree of branching and was less cytotoxic in a broad range of concentrations. As demonstrated by transmission electron microscopy the LMW-PEI formed only small aggregates which were efficiently taken up by different cells in the presence of serum, most likely by an endocytic pathway. LMW-PEI yielded transfection efficiencies measured via expression of the reporter gene luciferase which were up to two orders of magnitude higher than those obtained with HMW-PEI. The reporter gene expression was concentration dependent, but in contrast to lipofection independent of serum addition. Conclusions. The LMW-PEI described here is a new, highly efficient, and non-cytotoxic vector with a favorable efficiency/toxicity profile for gene therapeutic applications.