Pendant polymer:amino-β-cyclodextrin:siRNA guest:host nanoparticles as efficient vectors for gene silencing

A novel siRNA delivery vector has been developed, based on the self-assembly of monosubstituted cationic β-CD derivatives with a poly(vinyl alcohol)MW27kD (PVA) main-chain polymer bearing poly(ethylene glycol)MW2000 (PEG) and acid-labile cholesterol-modified (Chol) grafts through an acid-sensitive benzylidene acetal linkage. These components were investigated for their ability to form nanoparticles with siRNA using two different assembly schemes, involving either precomplexation of the pendant Chol-PVA-PEG polymer with the cationic β-CD derivatives before siRNA condensation or siRNA condensation with the cationic β-CD derivatives prior to addition of Chol-PVA-PEG to engage host:guest complexation. The pendant polymer:amino-β-CD:siRNA complexes were shown to form nanoparticles in the size range of 120-170 nm, with a slightly negative zeta potential. Cell viability studies in CHO-GFP cells shows that these materials have 10(3)-fold lower cytotoxicities than 25 kD bPEI, while maintaining gene-silencing efficiencies that are comparable to those of benchmark transfection reagents such as bPEI and Lipofectamine 2000. These results suggest that the degradable Chol-PVA-PEG polymer is able to self-assemble in the presence of siRNA and cationic-β-CD to form nanoparticles that are an effective and low-toxicity vehicle for delivering siRNA cargo to target cells.     

Effect of Cationic Lipid Nanoparticle Loaded siRNA with Stearylamine against Chikungunya Virus

Chikungunya is an infectious disease caused by mosquito-transmitted chikungunya virus (CHIKV). It was reported that NS1 and E2 siRNAs administration demonstrated CHIKV inhibition in in vitro as well as in vivo systems. Cationic lipids are promising for designing safe non-viral vectors and are beneficial in treating chikungunya. In this study, nanodelivery systems (hybrid polymeric/solid lipid nanoparticles) using cationic lipids (stearylamine, C9 lipid, and dioctadecylamine) and polymers (branched PEI-g-PEG -PEG) were prepared, characterized, and complexed with siRNA. The four developed delivery systems (F1, F2, F3, and F4) were assessed for stability and potential toxicities against CHIKV. In comparison to the other nanodelivery systems, F4 containing stearylamine (Octadecylamine; ODA), with an induced optimum cationic charge of 45.7 mV in the range of 152.1 nm, allowed maximum siRNA complexation, better stability, and higher transfection, with strong inhibition against the E2 and NS1 genes of CHIKV. The study concludes that cationic lipid-like ODA with ease of synthesis and characterization showed maximum complexation by structural condensation of siRNA owing to high transfection alone. Synergistic inhibition of CHIKV along with siRNA was demonstrated in both in vitro and in vivo models. Therefore, ODA-based cationic lipid nanoparticles can be explored as safe, potent, and efficient nonviral vectors overcoming siRNA in vivo complexities against chikungunya.     

Separation of superparamagnetic magnetite nanoparticles by capillary zone electrophoresis using non‐complexing and complexing electrolyte anions and tetramethylammonium as dispersing additive

Separations of bare superparamagnetic magnetite nanoparticles (BSPMNPs, approx. 11 nm diameter) was performed using non‐complexing (nitrate) and complexing (chloride, citrate and phosphate) electrolyte ions with additions of tetramethylammonium hydroxide (TMAOH), which is commonly applied to control the synthesis of stable iron oxides. The use of TMAOH as a background electrolyte (BGE) additive for capillary electrophoresis (CE) separations provided for the first time electropherograms of BSPMNPs exhibiting symmetrical and highly reproducible peaks, free of spurious spikes characteristic of nanoparticle clusters. Consequently, accurate determination of the electrophoretic effective mobility of BSPMNPs was possible, yielding a value of −3.345E‐08 m² V⁻¹ s⁻¹ (relative standard deviation (RSD) of 0.500%). The obtained mobilities of BSPMNPs in the presence of various electrolyte ions show that the degree of complexation with the surface of BSPMNPs follows the order chloride < citrate < phosphate, correlating with the stabilities of Fe(III) complexes with the respective anions. Finally, bare and carboxylated iron oxide nanoparticles were successfully separated in only 10 min using 10 mM Tris‐nitrate containing 20 mM of TMAOH as electrolyte. Our findings show that simple and rapid CE experiments are an excellent tool to characterise and monitor properties and interactions of iron oxide nanoparticles with other molecules for surface modification purposes.     

High-throughput screening of genetic mutations/polymorphisms by capillary electrophoresis

Genetic mutations/polymorphisms analyses play a great role in genetic and medical research, and clinical diagnosis. Most conventional methods for genetic assay are based on slab gel electrophoresis that is both labor-intensive and time-consuming. Recently, capillary electrophoresis (CE) has been used for genetic analysis instead of conventional slab gel electrophoresis. This technique can be automated and is characterized by short analysis time, small sample and reagents requirements, and high separation efficiency. CE has been successfully applied for mutation detection involving human tumor suppressor genes, oncogenes and disease-causing genes, and has shown a great potential for genetic mutation/polymorphism screening of large numbers of clinical samples. In this article, an overview of the fundamental aspects of mutation/polymorphism assay methods in combination with CE is given and some key applications are summarized.     

Determination of cyanides in electroplating solutions as Ni(CN)42– and analysis by capillary electrophoresis

A CE method was developed for the determination of total (free and weakly bound) cyanide in electroplating solutions based on the use of a cationic surfactant (TTAB) and complexation with Ni(II)-NH₃ solutions to Ni(CN)₄ ^2–. Both direct complexation and cyanide distillation combined with complexation were tested. Under optimized conditions, this method is time-saving compared to standard methods. Total cyanide determined by CE had detection limits (with respect to the initial sample concentration) of 0.5 μg/mL for direct complexation and 50 ng/mL for distillation combined with complexation. Total cyanide and cyanide not amenable by chlorination (CNAC) were determined in real samples from spent electroplating baths.     

Determination of cyanides in electroplating solutions as Ni(CN)42– and analysis by capillary electrophoresis

A CE method was developed for the determination of total (free and weakly bound) cyanide in electroplating solutions based on the use of a cationic surfactant (TTAB) and complexation with Ni(II)-NH₃ solutions to Ni(CN)₄ ^2–. Both direct complexation and cyanide distillation combined with complexation were tested. Under optimized conditions, this method is time-saving compared to standard methods. Total cyanide determined by CE had detection limits (with respect to the initial sample concentration) of 0.5 μg/mL for direct complexation and 50 ng/mL for distillation combined with complexation. Total cyanide and cyanide not amenable by chlorination (CNAC) were determined in real samples from spent electroplating baths. Received: 5 February 1998 / Revised: 26 July 1998 / Accepted: 1 August 1998     

Cover Picture: Electrophoresis 21'2010

Issue no. 21 is a regular issue with "Emphasis on Nucleic Acids" and comprises 17 contributions distributed over 4 distinct parts. Part I is on nucleic acids and has 8 research articles on various aspects of nucleic acid research including an automated instrument for human STR identification, a high resolution chip‐CE assay for rapid detection of gene mutations and amplifications, a fluorescence‐based sequence‐specific primer PCR, pulsed field CE for RNA separation, methylated DNA immunoprecipitation sequencing, PCR‐SSCP, DNA methylation, and analysis of SNPs. Part II has 4 research articles dealing with studies on protein and proteomics analysis, e.g., microchip IEF, CE analysis of microheterogeneous glutelin subunits in rice, protein precipitation methods and prefractionation of plasma for proteomic analysis. Part III has 2 contributions that describe the separation of the enantiomers of DL‐isocitric acid by CE and the analysis of trace degradation products of S‐adenosylmethionine, which are decarboxylated diastereomers, by CE. The last 3 articles in this issue (Part IV) are on novel methodologies for facile interfacing of CE with MS, electrokinetic chromatography for the determination of contaminants and impurities of heparin samples, and particle electrophoresis. As such, issue no. 21 is packed with the latest developments and innovations in the field. Featured articles include: An automated instrument for Human STR identification: design, characterization, and experimental validation (doi: 10.1002/elps.201000305 )) Miniature flowing atmospheric‐pressure afterglow ion source for facile interfacing of capillary electrophoresis with mass spectrometry (doi: 10.1002/elps.201000350 )) Capillary electrophoresis for analysis of microheterogeneous glutelin subunits in rice (Oryza sativa L.). (doi: 10.1002/elps.201000333 ))     

Gold and silver nanoparticles‐based laser desorption/ionization mass spectrometry method for detection and quantification of carboxylic acids

A comparison of ionization efficiency for gold and silver nanoparticles used as an active media of matrix‐less laser desorption/ionization (LDI) mass spectrometry (MS) methods was made for carboxylic acids including fatty acids. The matrix‐assisted laser desorption/ionization (MALDI)‐type targets containing monoisotopic cationic ¹⁰⁹Ag nanoparticles (¹⁰⁹AgNPs) and Au nanoparticles (AuNPs) were used for rapid MS measurements of 10 carboxylic acids of different chemical properties. Carboxylic acids were directly quantified in experiments with 10 000‐fold concentration change conditions ranging from 1 mg/ml to 100 ng/ml which equates to 1 μg to 100 pg of carboxylic acids per measurement spot.     

Continuous intact cell detection and viability determination by CE with dual‐wavelength detection

We introduce here a method for continuous intact cell detection and viability determination of individual trypan blue stained cells by CE with ultraviolet–visible dual‐wavelength detection. To avoid cell aggregation or damage during electrophoresis, cells after staining were fixed with 4% formaldehyde and were continuously introduced into the capillary by EOF. The absorbance of a cell at 590 nm was used to determine its viability. An absorbance of two milli‐absorbance unit at 590 nm was the clear cut‐off point for living and dead Hela cells in our experiments. Good viability correlation between the conventional trypan blue staining assay and our established CE method (correlation coefficient, R²=0.9623) was demonstrated by analysis of cell mixtures with varying proportions of living and dead cells. The CE method was also used to analyze the cytotoxicity of methylmercury, and the results were in good agreement with the trypan blue staining assay and 3‐(4,5‐dimethyl‐2‐thiazyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide methods. Compared with the 3‐(4,5‐dimethyl‐2‐thiazyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide method, our established CE method can be easily automated to report cell viability based on the state of individual cells. Tedious manual cell counting and human error due to investigator bias can be avoided by using this method.     

Direct Cytosolic Delivery of siRNA Using Nanoparticle‐Stabilized Nanocapsules

The use of nanoparticle‐stabilized nanocapsules (NPSCs) for the direct cytosolic delivery of siRNA is reported. In this approach, siRNA is complexed with cationic arginine‐functionalized gold nanoparticles by electrostatic interactions, with the resulting ensemble self‐assembled onto the surface of fatty acid nanodroplets to form a NPSC/siRNA nanocomplex. The complex rapidly delivers siRNA into the cytosol through membrane fusion, a mechanism supported by cellular uptake studies. Using destabilized green fluorescent protein (deGFP) as a target, 90 % knockdown was observed in HEK293 cells. Moreover, the delivery of siRNA targeting polo‐like kinase 1 (siPLK1) efficiently silenced PLK1 expression in cancer cells with concomitant cytotoxicity.     

Development of an imaged capillary isoelectric focusing method for characterizing the surface charge of mRNA lipid nanoparticle vaccines

Lipid nanoparticles (LNPs) have been employed for drug delivery in small molecules, siRNA, mRNA, and pDNA for both therapeutics and vaccines. Characterization of LNPs is challenging because they are heterogeneous mixtures of large complex particles. Many tools for particle size characterization, such as dynamic and static light scattering, have been applied as well as morphology analysis using electron microscopy. CE has been applied for the characterization of many different large particles such as liposomes, polymer, and viruses. However, there have been limited efforts to characterize the surface charge of LNPs and CIEF has not been explored for this type of particle. Typically, LNPs for delivery of oligonucleotides contain at least four different lipids, with at least one being an ionizable cationic lipid. Here, we describe the development of an imaged capillary isoelectric focusing method used to measure the surface charge (i.e., pI) of an LNP‐based mRNA vaccine. This method is capable of distinguishing the pI of LNPs manufactured with one or more different ionizable lipids for the purpose of confirming LNP identity in a manufacturing setting. Additionally, the method is quantitative and stability‐indicating making it suitable for both process and formulation development.     

Electrochemical derivatization‐capillary electrophoresis‐contactless conductivity detection: A versatile strategy for simultaneous determination of cationic,anionic, and neutral analytes

The simultaneous determination of cationic, anionic, and neutral analytes in a real sample was demonstrated by coupling electrochemical (EC) derivatization with counter‐EOF CE‐C⁴D. An EC flow cell was used to oxidize alcohols from an antiseptic mouthwash sample into carboxylic acids at a platinum electrode in acid medium. The carboxylates formed in the derivatization process and other sample ingredients, such as benzoate, saccharinate, and sodium ions, were separated in counter‐flow mode and detected in one run in Tris‐HCl buffer, pH 8.6. Fewer than 5 min were needed to complete each analysis with the automated flow system comprising solenoid pumps for the management of solutions. Insights into the electrochemistry of benzoic acid, present in the sample matrix, were also gained by EC‐CE‐C⁴D; more specifically, by applying potentials higher than 1.47 V to the platinum electrode, some formiate and minute amounts of salicylate were detected.     

Analysis of monoclonal antibody by a novel CE‐UV/MALDI‐MS interface

mAbs are highly complex proteins that present a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product‐ and time‐consuming. CE‐MS couplings, especially to MALDI, appear really attractive methods for the characterization of biological samples. In this work, we report the last instrumental development and performance of the first totally automated off‐line CE‐UV/MALDI‐MS/MS. This interface is based on the removal of the original UV cell of the CE apparatus, modification of the spotting device geometry, and creation of an integrated delivery matrix system. The performance of the method was evaluated with separation of five intact proteins and a tryptic digest mixture of nine proteins. Intact protein application shows the acquisition of electropherograms with high resolution and high repeatability. In the peptide mapping approach, a total number of 154 unique identified peptides were characterized using MS/MS spectra corresponding to average sequence coverage of 64.1%. Comparison with NanoLC/MALDI‐MS/MS showed complementarity at the peptide level with an increase of 42% when using CE/MALDI‐MS coupling. Finally, this work represents the first analysis of intact mAb charge variants by CZE using an MS detection. Moreover, using a peptide mapping approach CE‐UV/MALDI‐MS/MS fragmentation allowed 100% sequence coverage of the light chain and 92% of the heavy chain, and the separation of four major glycosylated peptides and their structural characterization.     

A family of single‐isomer,dicationic cyclodextrin chiral selectors for capillary electrophoresis: Mono‐6A‐ammonium‐6C‐butylimidazolium‐β‐cyclodextrin chlorides

The first member of the single‐isomer, dicationic cyclodextrin (CD) family, 6^A‐ammonium‐6^C‐butylimidazolium‐β‐cyclodextrin chlorides (AMBIMCD), has been synthesized, analytically characterized, and used to separate a variety of acidic enantiomers and amino acids by CE. Starting from mono‐6^A‐azido‐β‐cyclodextrin, the cationic imidazolium and ammonium moieties were subsequently introduced onto primary ring of β‐cyclodextrin via nucleophilic addition and Staudinger reaction. The analytically pure AC regio‐isomer CD was further obtained via column chromatography. This dicationic CD exhibited excellent enantioselectivities for selected analytes at concentration as low as 0.5 mM, which were even better than those of its mono‐imidazolium or ammonium‐substitued counterpart CDs at 10 equivalent concentrations. The effective mobilities of all studied analytes were found to decrease with the concentration of AMBIMCD. Inclusion complexation in combination with eletrostatic interactions seemed to account for the enhanced chiral discrimination process.     

Cover Picture: Electrophoresis 11'2011

Issue no. 11 is a regular issue consisting of 17 contributions distributed over 6 distinct parts: Part I is on fundamentals and methodologies containing 4 research articles dealing with electrokinetic transport through nanochannels, electroosmotic flow in polygonal ducts, analysis of complex samples by mass spectrometry using a sample pretreatment by preparative CITP, and CE analysis of inorganic cations in post‐blast residue extracts; Part II has 5 contributions on various aspects of proteomics and proteins including structural analysis of the recombinant therapeutic product rFVIII, electrophoretic characterization of proteins isolated by various extraction methods from ejaculated and capacitated boar sperms, CE separations of proteins using cationic poly(amidoamine) dendrimers, hemoglobin depletion in erythrocyte cytosolic samples, and peptide fractionation by acid pH SDS‐free electrophoresis; Part III is on separations of malignant cells, and particle capture in a droplet using dielectrophoresis; Part IV has two contributions on CEC separations; Part V consists of two papers on genetic variability and amplification; Part VI is on food analysis with one review article on CE applications in food analysis and one research paper on the determination of betaines in vegetable oils by CE‐MS. Thus, issue no. 11 covers many important subjects. Featured articles include: Electrokinetic transport through nanochannels (( 10.1002/elps.201000564 )) Interdigitated comb‐like electrodes for continuous separation of malignant cells from blood using dielectrophoresis (( 10.1002/elps.201000625 )) Experimental study of dielectrophoresis and liquid dielectrophoresis mechanisms for particle capture in a droplet (( 10.1002/elps.201000548 )) Thirty years of capillary electrophoresis in food analysis laboratories: Potential applications (( 10.1002/elps.201000541 ))     

Highly efficient approach for characterizing nanometer-sized gold particles by capillary electrophoresis

This paper demonstrates that capillary electrophoresis (CE) can be employed for characterizing the sizes of nanometer-scale gold particles. We characterized the gold nanoparticles by effecting CE separation using a buffer of SDS (70 mM) and 3-cyclohexylamino-1-propanesulfonic acid (CAPS; 10 mM) at pH 11.0 and an applied voltage of 18 kV and obtained a linear relationship (R² > 0.99) between electrophoretic mobilities and size for nanoparticles whose diameters fall in the regime from 5.0 ± 0.5 to 41.2 ± 3.3 nm; the relative standard deviations of these electrophoretic mobilities are <0.8%. We evaluated the feasibility of employing these separation conditions for the size characterization by of gold nanoparticle samples that were synthesized by a rapid microwave heating method. We confirmed that this CE method is a valid one for size characterization by comparing the results obtained by CE with those provided by scanning electron microscopy (SEM); a good correlation exists between these two techniques. Our results demonstrate that CE can be employed to accelerate the analysis of the sizes of nanomaterials.     

Study of Streptavidin-Modified Quantum Dots by Capillary Electrophoresis

Great boom of nanotechnologies impacts almost all areas of science and therefore detail understanding of the properties of nanomaterials as well as their interaction abilities is required. Surface modification and functionalization of nanoparticles is of a great interest due to the wide range of applications in the area of nanomedicine, nanobiology, and/or biochemistry. In this study, CdTe QDs were synthesized using microwave reactor and their surface was modified by streptavidin to ensure further suitability for bioconjugation with biotin-labelled oligonucleotides. For characterization of the synthesized QDs and for monitoring of the interaction with the oligonucleotide, capillary and gel electrophoresis was used. Moreover, complementary advantages of absorption (CE–UV) and laser-induced fluorescence detection (CE–LIF) were exploited. Comparison the electrophoretic mobilities obtained for streptavidin-modified QDs by CE–LIF (?9.87 × 10^?9 m²/V/s) and by CE–UV (?10.02 × 10^?9 m²/V/s) was in a good agreement enabling us to identify the peak of streptavidin-modified QDs in the CE–UV electropherogram containing also the peak of unreacted streptavidin. Subsequent conjugation of streptavidin-modified QDs with two model biotinylated oligonucleotides (BCL-2 and HBV) led to formation of the complex represented in the electropherograms as a very sharp peak. This peak height increased with time for 15.5 and 27 mAU using BCL-2 oligonucleotide and HBV oligonucleotide, respectively during 30 min interaction.     

Polycationic Amphiphilic Cyclodextrins for Gene Delivery: Synthesis and Effect of Structural Modifications on Plasmid DNA Complex Stability,Cytotoxicity, and Gene Expression

A molecular‐diversity‐oriented approach for the preparation of well‐defined polycationic amphiphilic cyclodextrins (paCDs) as gene‐delivery systems is reported. The synthetic strategy takes advantage of the differential reactivity of primary versus secondary hydroxyl groups on the CD torus to regioselectively decorate each rim with cationic elements and lipophilic tails, respectively. Both the charge density and the hydrophobic–hydrophilic balance can be finely tuned in a highly symmetrical architecture that is reminiscent of both cationic lipids and cationic polymers, the two most prominent types of nonviral gene vectors. The monodisperse nature of paCDs and the modularity of the synthetic scheme are particularly well suited for structure–activity relationship studies. Gel electrophoresis revealed that paCDs self‐assemble in the presence of plasmid DNA (pDNA) to provide homogeneous, stable nanoparticles (CDplexes) of 70–150 nm that fully protect pDNA from the environment. The transfection efficiency of the resulting CDplexes has been investigated in vitro on BNL‐CL2 and COS‐7 cell lines in the absence and presence of serum and found to be intimately dependent on architectural features. Facial amphiphilicity and the presence of a cluster of cationic and hydrogen‐bonding centers for cooperative and reversible complexation of the polyanionic DNA chain is crucial to attain high transgene expression levels with very low toxicity profiles. Further enhancement of gene expression, eventually overcoming that of polyplexes from commercial polyethyleneimine (PEI) polymers (22 kDa), is achieved by building up space‐oriented dendritic polycationic constructs.     

Podlike N‐Doped Carbon Nanotubes Encapsulating FeNi Alloy Nanoparticles: High‐Performance Counter Electrode Materials for Dye‐Sensitized Solar Cells

Podlike nitrogen‐doped carbon nanotubes encapsulating FeNi alloy nanoparticles (Pod(N)‐FeNi) were prepared by the direct pyrolysis of organometallic precursors. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and Tafel polarization measurements revealed their excellent electrocatalytic activities in the I^?/I₃^? redox reaction of dye‐sensitized solar cells (DSSCs). This is suggested to arise from the modification of the surface electronic properties of the carbon by the encapsulated metal alloy nanoparticles (NPs). Sequential scanning with EIS and CV further showed the high electrochemical stability of the Pod(N)‐FeNi composite. DSSCs with Pod(N)‐FeNi as the counter electrode (CE) presented a power conversion efficiency of 8.82 %, which is superior to that of the control device with sputtered Pt as the CE. The Pod(N)‐FeNi composite thus shows promise as an environmentally friendly, low‐cost, and highly efficient CE material for DSSCs.