Transfection agent

A plasmid-based transient expression system (InsectDirect system from EMD Biosciences Inc., USA www.emdbiosciences.com) will most probably greatly facilitate parallelization and automation for insect cell cultures. It generally gives lower yields, since expression is driven by an early baculoviral promoter, but it is possible to evaluate protein activity and expression level 24 h after transfection. It is also scalable to 1 L volume. The two main disadvantages, namely the large amount of transfection agent required and the limitation in scalability, can probably be overcome in future. 

The production of CLS by the melt dispersion technique is based on the melting of the lipid core material together with the lipophilic agent (i.e., phospholipids). Afterward, a warm aqueous solution is added to the molten material and is mixed by various methods (i.e., mechanical stirring, shaking, sonication, homogenization). Then the preparation is rapidly cooled until lipid solidification and the formation of particle dispersion. This method was used by Olbrich et al. [19] to produce the cationic solid lipid nanoparticles to use as novel transfection agent. 

Olbrich et al. [19] tested in vitro the cytotoxicity of the transfection agents, considering the viability of Cos-1 cell monolayers. Both the initial perturbation of cell integrity during the 4-h incubation and the influence on the cellular activity after 48 hours were assessed by measuring the release of lactate dehydrogenase and the mitochondrial conversion of the tetrazolium salt WST-1. Unmodified paraffin particles (SII-4) did not show cytotoxicity in the LDH (lactate dehydrogenase) release 2005 by CRC Press LLC... 

Another important aspect of transfection agents, especially for nonviral systems, was the efficiency/toxicity ratio. The CLS described by Olbrich et al. [19] showed only moderate transfection efficiencies compared with the established polymers... 

Philipp A, Zhao X, Tarcha P, Wagner E, Zintchenko A (2009) Hydrophobically modified oligoethylenimines as highly efficient transfection agents for siRNA delivery. Bioconjug Orem 20 2055-2061... 

Dendrimers bearing certain fluorescent molecules attached on their periphery have been shown to be environmentally sensitive probes for the presence of certain metal ions or to changes in pH (Balzani et al., 2000 Paola et al., 2005). In addition, PAMAM dendrimers modified with the relatively hydrophobic dye Oregon Green 488 were shown to be a more effective transfection agent for anti-sense oligonucleotides than the dendrimer alone-plus the complex could be tracked within the cell due to the fluorescence of the dye (Yoo and Juliano, 2000). 

Dendrimer-based MRI agents, 26 795-796 Dendrimer-based transfection agent,... 

Research Focus Development of high-efficiency biocompatible amidoamine dentrimeric transfection agents. 

Originality Although dentrimeric transfection agents are commercially available, the... 

Banerjee et al. (3) prepared hyperbranched dendrons consisting of polyethyle-neimine and 2-chloroethylamine hydrochloride for use as transfection agents. 

Component of liposomes Protective coating for liposomes Protein stabilizer DNA transfection agent Surfactant in intranasal or ocular delivery Increasing susceptibility of tumor cells to cytotoxic drugs Liver gene delivery... 

Walker S, et al. Cationic facial amphiphiles a promising class of transfection agents. Proc Natl Acad Sci USA 1996 93 1585-1590. 

Byk, G., Scherman, D., Schwartz, B. and Dubertret, C. (1997a) Lipopolyamines as transfection agents and pharmaceutical uses thereof. Patent Application, W0961774. 

Fig. 11 Transfection efficiency of carbamate-linked quaternary ammonium transfection agents (up-left) with chlorine or iodine counterions/pGL3 plasmid DNA complexes in COS-7 cell line [45]...

The effect of double bonds on both transfection and phase behavior is well illustrated by the pair C18 1/C10-EPC and C18 0/C10-EPC. These two lipids differ by one double bond only. However, the unsaturated lipid is over ten times more efficient as transfection agent than the saturated one [112]. The superior efficiency of C18 1/C10-EPC relative to C18 0/C10-EPC is also implied by the phases that evolve in membrane lipid formulations upon mixing with these two cationic lipids. A biomembrane-mimicking lipid formulation DOPC/DOPE/DOPS/Chol 45 20 20 15 remained lamellar in mixtures with C18 0/C10-EPC in contrast, the more efficient C18 1/C10-EPC induced a lamellar-nonlamellar phase conversion in this mixture, which was taking place at physiological temperature (Fig. 27). 

MacDonald RC, Rakhmanova VA, Choi KL et al (1999) O-Ethylphosphatidylcholine a metabolizable cationic phospholipid which is a serum-compatible DNA transfection agent. J Pharm Sci 88 896-904... 

Koynova R, Wang L, MacDonald RC (2006) An intracellular lamellar - nonlamellar phase transition rationalizes the superior performance of some cationic lipid transfection agents. Proc Nad Acad Sci USA 103 14373-14378... 

Besides generation number and degree of flexibility, hyperbranched PAMAM can be synthesized with different chemical properties [37,43, 56, 58, 80,114,116], The biotechnology company QIAGEN offers two commercially available hyperbranched PAMAM dendrimers Superfect and Polyfect as in vitro transfections agents, which have been successfully used in many laboratories. 

Fig. 8 (a) Influence of dendrimer generation on transfection efficiency, (b) Influence of dendrimer activation on transfection efficiency. The different bar colors represent different amounts of transfection agent used. Reproduced with permission from [117]. Copyright 2002 Elsevier... 

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