Protein Expression Knockdown in Cancer Cells Induced by a Gemini Cationic Lipid Nanovector with Histidine-Based Polar Heads.

A histidine-based gemini cationic lipid, which had already demonstrated its efficiency as a plasmid DNA (pDNA) nanocarrier, has been used in this work to transfect a small interfering RNA (siRNA) into cancer cells. In combination with the helper lipid monoolein glycerol (MOG), the cationic lipid was used as an antiGFP-siRNA nanovector in a multidisciplinary study. Initially, a biophysical characterization by zeta potential (ζ) and agarose gel electrophoresis experiments was performed to determine the lipid effective charge and confirm siRNA compaction. The lipoplexes formed were arranged in Lα lamellar lyotropic liquid crystal phases with a cluster-type morphology, as cryo-transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS) studies revealed. Additionally, in vitro experiments confirmed the high gene knockdown efficiency of the lipid-based nanovehicle as detected by flow cytometry (FC) and epifluorescence microscopy, even better than that of Lipofectamine2000*, the transfecting reagent commonly used as a positive control. Cytotoxicity assays indicated that the nanovector is non-toxic to cells. Finally, using nano-liquid chromatography tandem mass spectrometry (nanoLC-MS/MS), apolipoprotein A-I and A-II followed by serum albumin were identified as the proteins with higher affinity for the surface of the lipoplexes. This fact could be beyond the remarkable silencing activity of the histidine-based lipid nanocarrier herein presented.

Biocompatible Nanovector of siRNA Consisting of Arginine-Based Cationic Lipid for Gene Knockdown in Cancer Cells.

Despite the use of small interfering RNAs (siRNAs) as therapeutic agents through the knockdown expression of pathogenic proteins, transportation and delivery of such siRNAs into cells continue to be under investigation. Within nonviral vectors, cationic lipids that include amino acid residues in their structures, and that have already demonstrated their suitability as plasmid DNA nanocarriers, may be also considered as potential siRNA vehicles. A double-chain cationic lipid based on the amino acid arginine mixed with a helper lipid has been the object of this biophysical study. First, ζ-potential measurements and agarose gel electrophoresis experiments confirmed the siRNA compaction, while small-angle X-ray scattering analysis (SAXS) revealed the structural pattern of the lipoplexes. Two bicontinuous cubic phases were found to coexist: the double-gyroid phase (QIIG) and the double-diamond phase (QIID), with Pn3m and Ia3d as crystallographic space groups, respectively; the siRNA is known to be located inside their bicontinuous aqueous channels. Second, in vitro studies in HeLa-green fluorescent protein (GFP) and T731-GFP cell lines (modified for GFP overexpression) showed moderate to high gene knockdown levels (determined by flow cytometry and epifluorescence microscopy) with remarkable cell viabilities (CCK-8 assay). Finally, nano-liquid chromatography/mass spectrometry (nanoLC-MS/MS) was used to identify the nature of the proteins adhered to the surface of the lipoplexes after incubation with human serum, simulating their behavior in biological fluids. The abundant presence of lipoproteins and serum albumin in such protein corona, together with the coexistence of the bicontinuous cubic phases, may be behind the remarkable silencing activity of these lipoplexes. The results reported herein show that the use of amino-acid-based cationic lipids mixed with a suitable helper lipid, which have already provided good results as DNA plasmid nanocarriers in cellular transfection processes, may also be a biocompatible option, and so far little investigated, in gene silencing in vitro strategies.

Evaluation of the detection of biomolecules in capillary electrophoresis by contactless conductivity measurement.

The sensitivity of contactless conductivity detection to amino acids, peptides and proteins in CE was studied for BGE solutions of different pH values. The LOD and analytical characteristics were compared for acidic and basic conditions and better results were in most cases found for buffers of low pH values. Linear dynamic ranges varied between two orders of magnitude for amino acids and peptides and three orders of magnitude for larger proteins. The concentration detection limits were found to be between 1.2 and 7.5 microM for the amino acids tested and for the larger molecules they varied between 2.6 microM for leucine enkephalin and 0.2 microM for HSA when using a buffer at pH 2.1.

Determination of biochemical species on electrophoresis chips with an external contactless conductivity detector.

Contactless conductivity measurements were found to be suitable for the direct detection, i.e., without needing any labels, of a range of biochemically relevant species, namely amino acids, peptides, proteins, immunoglobulin, and DNA. It was also possible to monitor the products of the enzymatic digestion of HSA with pepsin. Detection was carried out on bare electrophoresis chips made from poly(methyl methacrylate) by probing the conductivity in the channel with a pair of external electrodes, which are fixed on the chip holder. Separation efficiencies up to 15,000 plates could be obtained and LODs are in the low muM-range, except for immunoglobulin G (IgG) which could be determined down to 0.4 nM. Linear dynamic ranges of two to three orders of magnitude were obtained for the peptides as examples.

Contactless conductivity detection of selected organic ions in on-chip electrophoresis.

The detection of underivatized anionic sulfonates, carboxylates, amino acids, sugars, and artificial sweeteners, and of cationic dopamine, ephedrine, and metanephrine in microfabricated electrophoresis devices is demonstrated. This was achieved by high-voltage contactless conductivity measurements with external electrodes. Poly(methyl methacrylate) chips with thin covers to enable sensitive contactless detection were used for most determinations but glass microchips had to be employed for amino acids and sugars. The plastic chips were found not be stable in the alkaline media required to render those two classes of species in the ionic form amenable for separation and detection. The reproducibility of peak area measurements was about 1% or better and the detection limits ranged between 1 and 30 microM for the different compounds examined.

Detection of human immunoglobulin in microchip and conventional capillary electrophoresis with contactless conductivity measurements.

The detection of human immunoglobulin M (IgM) was performed using capacitively coupled contactless conductivity detection (CCD) in electrophoresis carried out in conventional capillaries as well as on glass and poly(meth-yl methacrylate) (PMMA) microdevices. Also achieved was the analyses of IgG (an anti-human IgM) and the complex formed in the reaction between the two immunoreagents. It is demonstrated that CCD is a powerful tool suitable not only for the detection of antibodies but also for monitoring an immunological interaction. Conductivity measurements allow the direct determination of immunoreagents, and it is advantageous, since no labels are required. The immunoglobulin IgM has been taken as model analyte. The reproducibility of the analytical signal (RSD = 1%), sensitivity and limits of detection obtained for IgM (0.15 ng/mL in conventional capillaries and 34 ng/mL in microchips) are comparable to those previously obtained with amperometric detection. The immunological reaction was performed either in conventional microtiter plates as used in ELISA or in situ on the glass chip.

The use of gold bands for flow immunoelectrochemical devices.

Gold bands sputtered over a polymeric material, Kapton, have been employed not only for electrochemical detection but also for the development of enzyme immunoassays in a flow system. The immunological interactions on bands acting as reactors are considered for a model analyte, IgM. Different formats of flow immunoassays, competitive and non-competitive, have been checked. Compared with previous results, automation gives rise to in a reduction in analysis time and in reagent consumption. Lower limits of detection are also obtained. Detection, which is also carried out in the flow system, is based on the oxidation of naphthol, the product of the enzymatic hydrolysis of naphthyl-phosphate.

Voltammetric and flow amperometric methods for the determination of melatonin in pharmaceuticals.

Melatonin can be sensitively detected in pharmaceuticals by two different and simple electrochemical methods: cyclic voltammetry (CV) and amperometric detection in a flow injection analysis system (FIA-ED). An adequate pre-treatment of the carbon paste electrode in the first case and the employ of a high flow rate in the second one were the key for obtaining a very good reproducibility (R.S.D. values of 1.5 (n=10) and 1.3% (n=20), respectively). Low limits of detection were achieved and with the coupling of a flow system a linear dynamic range of three orders of magnitude (from 10(-8) to 10(-5) M) was obtained. Both methods were applied to the determination of melatonin in pharmaceuticals. In order to best validate these methodologies a fluorescent procedure was developed to contrast the results. As no interferences from the matrix were found the employ of a separation technique is not necessary. In this way the procedure is fastened and simplified. Moreover, the low price, ease of handling, possibility of automation and high sample throughput are important advantages that convert the flow methodology in an attractive alternative for quality control of pharmaceuticals.

Gold bands as a suitable surface for enzyme immunoassays.

Gold bands sputtered over a polymeric material, Kapton, are employed for the development of enzyme immunoassays. The immunological interaction takes place between human IgM and alkaline phosphatase (AP) conjugated anti-IgM. The model analyte (IgM) could be determined following a non-competitive design in the range of 0.05-5 ppm, with a limit of detection of 50 ppb. After the interaction, gold bands are sequentially inserted in a flow system and the extension of the reaction is followed through the enzymatic hydrolysis of naphthylphosphate, AP substrate. The product, naphthol, is oxidised to naphtoquinone in the gold band of the flow cell that constitutes the detector. Parameters affecting the interaction are studied and calibration curves are performed. The reproducibility between different bands (RSD=4%, n=5) and possibilities of regeneration are also detailed.