Layered double hydroxides : synthesis and application in gene delivery to mammalian cells in culture.
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Layered double hydroxides (LDHs) or hydrotalcite-like compounds (HTLcs) are classified as anionic clays in which their structure is based upon brucite and are represented by the following general chemical formula: [MII1-xMIII x(OH)2]x+(An-)x/y.yH2O where MII and MIII represent various possible divalent cations, e.g., Mg2+, Zn2+, Ni2+, Co2+ and Fe2+ and trivalent cations, e.g., Al3+, Fe3+ and Cr3+ respectively. The value x is equal to the stoichiometric ratio of MIII/(MII+MIII) and An- represents exchangeable anions such as CO32-, Cl- and SO42-. It is these exchangeable interlayer anions, which make layered double hydroxide compounds excellent carriers of negatively charged or anionic containing biomolecules, such as DNA and hence can be exploited in their use in gene therapy. In this study, a variety of Mg-Al hydrotalcites (HTs), Zn-Al, Zn-Fe and Mg-Fe LDHs were synthesized using co-precipitation. The synthesized compounds were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, Inductively coupled plasma-optical emission spectroscopy (ICP-OES), Transmission electron microscopy (TEM), Scanning electron microscopy (SEM) and Scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDX). XRD patterns for the synthesized HTs and LDHs exhibited characteristic features indicative of an ordered layered material. Elemental analysis of the compounds revealed a measured value of x in the range of 0.25-0.33 for Mg-Al HTs and Zn-Fe LDHs, 0.11-0.16 for Zn-Al LDHs and 0.55-0.58 for Mg-Fe LDHs. FTIR and Raman spectroscopy confirmed the presence of characteristic functional groups and interlayer anions. From electron microscopy, the compounds exhibited classical morphologies typical of HT and LDH compounds and had a lateral size range of 200-300 nm. These compounds were studied in their ability to bind DNA with the use of a gel retardation or band shift assay. This assay confirmed that these compounds are indeed able to bind DNA. The binding mechanism of DNA to the HT and LDH compounds was evaluated and plausible mechanisms were proposed. Furthermore, nuclease digestion assays were carried out in order to evaluate the potential protecting ability that these compounds afford towards the bound DNA in the presence of serum. It was observed that all compounds protected most of the bound DNA. The cytotoxicity of the compounds was evaluated in the HEK293, HepG2 and HeLa mammalian cell lines using the MTS (3-(4,5-dimethylthiazol-2yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salts) assay with a concentration range of 20-100 μg of respective HT/LDH compound. For most of the compounds, cell viability was observed in excess of 80 %. Finally, transfection studies were carried out utilizing green fluorescent protein (GFP) analysis and luciferase gene expression using the same mammalian cells in culture. It was noted that all HTs and LDHs were able to release DNA in a controlled prolonged manner over a period of three days. Green fluorescent protein gene expression commenced after 27 hours and reached a maximum at 72 hours. Efficient luciferase gene expression was observed with luciferase activities for DNA: HTs ranging from 0.05 x 106 - 2.0 x 106 RLU / mg protein and DNA: LDHs ranging from 0.05 x 106 - 16.7 x 106 RLU / mg protein. Highest luciferase activity was recorded as 16.7 x 106 RLU / mg protein.