Elsevier

Journal of Controlled Release

Volume 141, Issue 2, 25 January 2010, Pages 110-127
Journal of Controlled Release

Review
How to study dendriplexes II: Transfection and cytotoxicity

https://doi.org/10.1016/j.jconrel.2009.09.030Get rights and content

Abstract

This paper reviews different techniques for analyzing the transfection efficiencies and cytotoxicities of dendriplexes–complexes of nucleic acids with dendrimers. Analysis shows that three plasmids are mainly used in transfection experiments: plasmid DNA encoding luciferase from the firefly Photinus pyralis, β-galactosidase, or green fluorescent protein. The effective charge ratio of transfection does not directly correlate with the charge ratio obtained from gel electrophoresis, zeta-potential or ethidium bromide intercalation data. The most popular cells for transfection studies are human embryonic kidney cells (HEK293), mouse embryonic cells (NIH/3T3), SV40 transformed monkey kidney fibroblasts (COS-7) and human epithelioid cervical carcinoma cells (HeLa). Cellular uptake is estimated using fluorescently-labeled dendrimers or nucleic acids. Transfection efficiency is measured by the luciferase reporter assay for luciferase, X-Gal staining or β-galactosidase assay for β-galactosidase, and confocal microscopy for green fluorescent protein. Cytotoxicity is determined by the MTT test and lactate dehydrogenase assays. On the basis of the papers reviewed, a standard essential set of techniques for characterizing dendriplexes was constructed: (1) analysis of size and shape of dendriplexes in dried/frozen state by electron or atomic force microscopy; (2) analysis of charge/molar ratio of complexes by gel electrophoresis or ethidium bromide intercalation assay or zeta-potential measurement; (3) analysis of hydrodynamic diameter of dendriplexes in solution by dynamic light scattering. For the evaluation of transfection efficiency the essential techniques are (4) luciferase reporter assay, β-galactosidase assay or green fluorescent protein microscopy, and (5) cytotoxicity by the MTT test. All these tests allow the transfection efficiencies and cytotoxicities of different kinds of dendrimers to be compared.

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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