ReviewHow to study dendriplexes II: Transfection and cytotoxicity
Introduction
Dendrimers are a new class of polymers with a well-defined molecular structure [1], [2], [3], [4], [5], [6]. Because of specific synthesis conditions, they combine defined composition and monodispersivity with high molecular mass, so they have numerous interesting physical and chemical properties. They possess many functional end groups, which are responsible for high solubility and reactivity, and empty internal cavities [1], [2], [3], [4], [5], [6]. These properties make dendrimers suitable for targeting of nucleic acids and short oligodeoxynucleotides (ODN) [7], [8]. Such complexes have been called dendriplexes by analogy with polyplexes (polymer/nucleic acid complexes) and lipoplexes (liposome/nucleic acid complexes).
In a previous paper [9] we reviewed the techniques for physical–chemical characterization of dendriplexes [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], [45], [46], [47], [48], [49], [50], [51], [52], [53], [54], [55], [56], [57], [58], [59], [60], [61], [62], [63], [64], [65], [66], [67], [68], [69], [70], [71], [72], [73], [74], [75], [76], [77], [78], [79], [80], [81], [82], [83], [84], [85], [86], [87], [88], [89]. In the present review we have tried to systemize the techniques for evaluating the transfection efficiencies and cytotoxicities of dendriplexes.
Section snippets
Information
Several reviews are devoted to the use of dendrimers in gene transfection [7], [8], [88], [89]. Transfection experiments can be categorized according to two different approaches: (1) the delivery of plasmid DNA to express a gene of interest under the control of a suitable promoter, which will result in the increased production of a protein; (2) the silencing of a gene using oligomeric genetic material such as antisense ODN, siRNA or a DNAzyme, which will lead to a reduction of target/protein
Information
The cell lines and animals used in dendrimer-mediated transfection in vitro and in vivo are listed in Table 3, Table 4.
Table 3 shows that the most popular cells for transfection studies are human embryonic kidney cells (HEK293) [19], [26], [28], [29], [47], [50], [53], [68], [71], [90], [115], [121], [138], [139], human epithelioid cervical carcinoma cells (HeLa) [10], [23], [51], [53], [68], [107], [113], [118], [121], [129], [133], SV40 transformed monkey kidney fibroblasts (COS-7) [11], [19]
Information
Complexing dendrimers with nucleic acids is based mainly on the interaction between the anionic phosphate groups of the DNA and cationic groups on the dendrimers [7], [8], [88], [89]. These complexes are colloidally stable if they are charged, which means that the cationic dendrimer has to be present in excess. Moreover, the positive charge enables them to interact with cell receptors. This is useful for experiments in vitro, but in vivo it can lead to non-specific binding of complexes and
Cellular uptake
Cellular uptake can be estimated by radioactive or fluorescent labeling.
Information
Firefly luciferase is widely used as a reporter for the following reasons: (1) reporter activity is available immediately upon translation since the protein does not require post-translational processing; (2) the assay is very sensitive because its light production has the highest quantum efficiency known for any chemiluminescent reaction and no background luminescence is found in the host cells or the assay chemistry; (3) the assay is rapid, requiring only a few seconds per sample [10], [11],
Information
Mitochondrial dehydrogenase activities may be determined by the MTT test. The tetrazolium salt MTT (a water-soluble yellow dye) is converted by live cells into blue, insoluble formazan crystals, which then have to be dissolved in a suitable extraction mixture and measured spectrophotometrically [149].
Procedure
In a typical experiment [10], cells were plated at a density of about 5 × 104 cells per well in 300 µl medium in 96-well trays. After an overnight culture at 37 °C in a 5% CO2 humidified atmosphere,
Conclusions
In most transfection experiments, three plasmids are used: plasmid DNA encoding luciferase from the firefly P. pyralis, β-galactosidase or green fluorescent protein. The effective charge ratio of transfection does not correlate with those obtained from gel electrophoresis, zeta-potential or ethidium bromide intercalation assay data. The most popular cells for transfection studies are human embryonic kidney cells (HEK293), mouse embryonic cells (NIH/3T3), SV40 transformed monkey kidney
Acknowledgement
This work has been supported by grant from the ERA-NET NAN2007-31198-E.
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2019, Colloids and Surfaces B: BiointerfacesCitation Excerpt :Synthesis of multifunctional conjugates on the basis of PAMAM dendrimers produces safe and biocompatible nano-carriers with the potential of having prolonged circulation half-life, bioresponsiveness and target-specific degradability [8,9,13,14]. Generation 4 (G4) of cationic PAMAM dendrimers proved to be the most significant carrier for DNA and RNA [8,9,13,14]. Previously we synthesized new cationic piperidine-based phosphorous dendrimers [15], which had excellent binding ability and could transfect siRNAs, providing 80–100% siRNA uptake by HeLa cells in serum-containing medium, whereas the widespread transfection agent, Lipofectamine, showed only ˜40% uptake, with cationic PAMAM dendrimers of 3–4 generations giving only ˜60–80% uptake (data not presented) [15].