Accuracy estimation of multiangle light scattering detectors utilized for polydisperse particle characterization with field-flow fractionation techniques: a simulation study

The coupling of field-flow fractionation (FFF) and multiangle light scattering (MAIS) detectors is complementary in that the MALS system allows particle characterization when a narrow dispersity particle population is present in the detector. The fractionation process provides this narrow dispersity. Utilizing discrete particle simulations of FFF and optical calculations based on both the Mie theory of particle scattering and Rayleigh-Gans-Debye (RGD) scattering theory, the extent of polydispersity that can be tolerated for accurate particle quantitation is explored. It is found that flow, electrical, and sedimentation FFF provide adequate separation for accurate particle quantitation by MALS. The Mie theory is more accurate than the RGD theory, which is known to deviate at higher particle size. Low error in the measurement of mean diameters is found when only the particle diameter is of interest. It is shown that the reconstruction of the particle size distribution from time slice data is distorted due to errors in concentration, which result from finite polydispersity and other effects. A number of procedures are evaluated in restoring the size distribution to higher accuracy. None of these procedures is deemed of general purpose and none of these is reliable. The best results are obtained when fractionation is conducted under the minimal possible outlet polydispersity and when steric effects are minimized. In addition, best results are had for inherently narrow dispersity colloidal materials.     

A novel cationic liposome formulation for efficient gene delivery via a pulmonary route

The clinical success of gene therapy for lung cancer is not only dependent on efficient gene carriers but also on a suitable delivery route. A pulmonary delivery route can directly deliver gene vectors to the lung which is more efficient than a systemic delivery route. For gene carriers, cationic liposomes have recently emerged as leading non-viral vectors in worldwide gene therapy clinical trials. However, cytotoxic effects or apoptosis are often observed which is mostly dependent on the cationic lipid used. Therefore, an efficient and safe cationic lipid, 6-lauroxyhexyl lysinate (LHLN), previously synthesized by our group was first used to prepare cationic liposomes. Physicochemical and biological properties of LHLN-liposomes were investigated. LHLN-liposome/DNA complexes showed positive surface charge, spherical morphology, a relatively narrow particle size distribution and strong DNA binding capability. Compared with Lipofectamine2000, the new cationic liposome formulation using LHLN exhibited not only lower cytotoxicity (P < 0.05) but also similar transfection efficiency in A549 and HepG2 lung cancer cells for in vitro tests. When administered by intratracheal instillation into rat lungs for in vivo evaluation, LHLN-liposome/DNA complexes exhibited higher pulmonary gene transfection efficiency than Lipofectamine2000/DNA complexes (P < 0.05). These results suggested that LHLN-liposomes may have great potential for efficient pulmonary gene delivery.     

Determination of the size of water-soluble nanoparticles and quantum dots by field-flow fractionation

Field flow fractionation (FFF) technique is used to determine the size of water-soluble Au, ZnS, ZnS-Mn2+ nanoparticles, and CdSe, CdSe-DNA quantum dots (QDs). The results of the FFF measurements are compared with the particle size analysis using conventional techniques like scanning electron microscopy (SEM), transmission electron microscopy (TEM), and dynamic light scattering (DLS) studies. Water-soluble gold nanoparticles (AuNPs) stabilized by mercaptosuccinic acid (MSA) as the ligand when analyzed by the SEM and DLS showed evidence of extensive aggregation, preventing an accurate determination of the average particle size. The TEM analyses without staining offered a facile measurement of the nanoparticle core but average particle size determination required analysis of the TEM image using image analysis software. On the other hand the FFF is seemingly a convenient and easy method for the determination of the average particle size of the AuNPs. In case of the ZnS and ZnS-Mn2+ nanoparticles with mercaptopropionic acid (MPA) as the capping agent severe aggregation prevented accurate estimation of particle sizes even by the high resolution TEM (HRTEM), where as the size determination by the FFF was very facile. Analysis of the CdSe-DNA conjugate by the TEM was difficult as the sample got damaged upon exposure to the electron beam. The FFF cross-flow condition is apparently noninvasive and hence the technique was very effective in characterizing the CdSe-DNA QDs. Furthermore, using this simple technique it was possible to fractionate a sample of the AuNPs. The FFF measurement of water-soluble nanoparticles is an excellent complement to characterization of such particles by the conventional tools.     

Structural characterization of novel gemini non-viral DNA delivery systems for cutaneous gene therapy

The structural and physicochemical properties of novel cationic lipid-based DNA complexes have been investigated for the purpose of designing micro/nano-scale self-assembling delivery systems for cutaneous gene therapy. DNA/gemini surfactant (spacer n?=?3–16; chain m?=?12 or 16) complexes (1?:?10 charge ratio), with or without dioleoylphosphatidyl-ethanolamine (DOPE), designed for cellular transfection, were generally in the range of 100–200?nm as demonstrated by atomic force microscopy and particle size analysis. Small-angle X-ray scattering measurements indicated that the DNA/gemini complexes lacked long-range order, whereas DNA/gemini/DOPE complexes exhibited lamellar and polymorphic phases other than hexagonal. Correlation studies using transfection efficiency data in PAM 212 keratinocytes and in vitro skin absorption indicated that formulations containing gemini surfactants having the ability to induce structures other than lamellar in the resulting complexes, generally exhibited greater transfection activity and cutaneous absorption.     

Determination of mean diameter and particle size distribution of acrylate latex using flow field-flow fractionation, photon correlation spectroscopy, and electron microscopy

Flow field-flow fractionation (flow FFF) was employed to determine the mean diameter and the size distribution of acrylate latex materials having diameters ranging from 0.05 to 1 μm. Mean diameters of the samples determined by flow FFF are in good agreement with those obtained from photon correlation spectroscopy (PCS). Scanning electron microscopy (SEM) yielded a mean diameter that is about 20% lower than those obtained from flow FFF or PCS, probably due to the shrinkage of particles during sample drying and high-vacuum measurements. It was found that flow FFF is particularly useful for the determination of particle size distributions of latex materials having broad size distributions. Flow FFF separates particles according to their sizes and yields an elution curve that directly represents the particle size distribution of the sample. In PCS, measurements had to be repeated at more than one scattering angle to obtain an accurate mean diameter for the latex having a broad size distribution. Flow FFF was fast (less than 12 min of run time) and showed an excellent repeatability in measuring the mean diameter with ±5% relative error.     

Bacteria sorting by field-flow fractionation. Application to whole-cell Escherichia coil vaccine strains

Sorting and quantification of deactivated bacteria is an important way of quality control for whole-cell bacterial vaccines. In general, surface features of deactivated bacteria used for whole-cell bacterial vaccines affect the immunoresponse to bacteria-associated antigens. Enumeration of bacteria is also an important process development parameter for these vaccines. Field-flow fractionation (FFF) was previously applied to the separation of bacteria. For the first time, FFF is used for sorting bacteria strains of the same species on the basis of differences in bacterial membrane characteristics. Two FFF techniques, gravitational FFF (GrFFF) and asymmetrical flow FFF (AsFIFFF), are shown to be able to fractionate, distinguish, and quantify different deactivated Escherichia coli strains used for vaccines. E. coli can differ in the presence of fimbriae on the bacterial membrane. Fimbriae affect E. coli pathology and thus the use of E. coli for vaccines. GrFFF and AsFIFFF are able to fractionate fimbriated/ nonfimbriated cells in mixtures of different strains. While GrFFF is characterized by low cost and simplicity, As-FIFFF shows a higher performance in size fractionation with a high-speed separation. Coupled, on-line UV/visible turbidimetry yields the relative numbers of fractionated cells and sample recovery. Scanning electron microscopy and quasi-elastic light scattering are employed as uncorrelated techniques for size and morphology analysis of the E. coli strains.     

Ultrahigh molar mass component detected in ethylhydroxyethyl cellulose by asymmetrical flow field-flow fractionation coupled to multiangle light scattering.

Asymmetrical flow field-flow fractionation (flow FFF) was connected to multiangle light scattering (MALS) and refractive index (RI) detectors for characterization of the molar mass distribution and molecular radius of a cellulose derivative, ethylhydroxyethyl cellulose (EHEC). Experimental conditions were optimized to allow study of a wide range of molar mass including even ultrahigh molar mass (UHM) components. The weight-average molar mass was 3.1 x 10(5) g x mol(-1) representing a very broad range (of molar mass) from 4.0 x 10(4) to 10(7) g x mol(-1), which corresponds to from <20 to 200 nm rms radius. The light scattering signal showed the presence of an UHM component, possibly an aggregate of extreme size, i.e., approximately 10(8) g x mol(-1) with a hydrodynamic diameter of 0.35 microm. Careful choice of the pore size in in-line filters is necessary in order to minimize MALS detector noise without removing the UHM component. Flow FFF-MALS-RI was demonstrated to be uniquely suited to detect the presence of UHM components.     

Dual-field and flow-programmed lift hyperlayer field-flow fractionation.

Field and flow programming and their combination, dual programming, are shown to extend the particle size range to which a single flow/hyperlayer field-flow fractionation (FFF) run is applicable to approximately 1-50 microns. The rationale for programming flow/hyperlayer FFF (or other forms of lift hyperlayer FFF) is to expand the diameter range of micron size particles that can be resolved in a single run. By contrast, the reason for programming normal-mode FFF, the only kind of programming previously realized in FFF, is to reduce the analysis time of submicron particle samples of considerable size variability. These differences are explained in detail in relationship to the basic mechanisms governing retention in normal, steric, and lift hyperlayer FFF. Experiments are described in which field, flow, and dual programming are used to expand the accessible diameter range of flow/hyperlayer FFF. An example is shown in which 11 sizes of latex microspheres in the 2-48-microns diameter range are separated by dual programming in 11 min.     

Amphiphilic star block copolymers as gene carrier Part I: Synthesis via ATRP using calix[4]resorcinarene-based initiators and characterization

In this work, a cationic star polymer [poly(2-dimethylamino)ethyl methacrylate (PDMAEMA)] was prepared via atom transfer radical polymerization (ATRP), using brominated calix[4]resorcinarene as an initiator. Hydrophobic moieties, methyl methacrylate (MMA) and butyl acrylate (BA), were further incorporated via “one-pot” method. Well-defined eight-armed star block copolymers bearing hydrophilic blocks inside and hydrophobic blocks outside were synthesized. The molecular weight, particle size, electrophoretic mobility and apparent charge density were determined by gel permeation chromatography (GPC), dynamic light scattering (DLS), phase analysis light scattering (PALS) and colloidal titration, respectively. The zeta potentials and apparent charge densities of the products exhibited the characteristics of polyelectrolyte. The incorporation of hydrophobic moieties generated electrostatic screening effect. The as-synthesized amphiphilic star copolymer is promising as a thermo-sensitive gene carrier for gene therapy.     

Polydispersity characterization of lipid nanoparticles for siRNA delivery using multiple detection size-exclusion chromatography

The development of lipid nanoparticle (LNP) based small interfering RNA (siRNA) therapeutics presents unique pharmaceutical and regulatory challenges. In contrast to small molecule drugs that are highly pure and well-defined, LNP drug products can exhibit heterogeneity in size, composition, surface property, or morphology. The potential for batch heterogeneity introduces a complexity that must be confronted in order to successfully develop and ensure quality in LNP pharmaceuticals. Currently, there is a lack of scientific knowledge in the heterogeneity of LNPs as well as high-resolution techniques that permit this evaluation. This article reports a size-exclusion chromatography (SEC) method that permits the high-resolution analysis of LNP size distribution in its native solution condition. When coupled with multiple detection systems including UV-vis, multi-angle light scattering, and refractive index, on-line characterization of the distributions in size, molecular weight, and siRNA cargo loading of LNPs could be achieved. Six LNPs with sizes in the rang of 60-140 nm were evaluated and it was found that the SEC separation is efficient, highly reproducible, and can be broadly applied to a diverse range of LNPs. A comparison between the current SEC method and asymmetric field flow fractionation (FFF) shows that the current method provides similar size distribution results on LNPs compared to FFF. Two representative LNPs with similar bulk properties were evaluated in-depth using the SEC method along with two other sizing techniques-dynamic light scattering and cryo-TEM. Profound differences in batch polydispersity were observed between them. Despite the similarity in the particle assembly process, it was found that one LNP (A) possessed a narrow size and molecular weight distribution while the other (B) was polydisperse. The present results suggest that LNP drug products are highly complex and diverse in nature, and care should be taken in examining and understanding them to ensure quality and consistency. The method developed here can not only serve as a method for understanding LNP product property, permitting control on product quality, but also could serve as a potential manufacturing method for product purification. Understandings obtained in this work can help to facilitate the development of LNPs as a well-defined pharmaceutical product.     

Light scattering and absorption caused by bacterial activity in water

There is a growing class of elementary particle detectors, large-water ?erenkov detectors, that have a body of water (thousands of tons) as a sensitive medium. Particles are detected when they interact with the water and produce ?erenkov light, so detection efficiency relies on the transparency of the water. These detectors are active typically for many years, so biological activity (primarily bacterial growth) is one of the means by which the transparency of the water may be reduced. We present the results of a measurement of light scattering and absorption from a population of Escherichia coli in water, which is used as a model for bacteria in general. One can separate the scattering and absorption by varying the refractive index of the medium by using a solute of high molecular weight. We show that the results can be understood simply in terms of light scattering from small spheres (radius ≈ wavelength) with an effective refractive index, n(b), plus a small amount of absorption in the ultraviolet. We compare his scattering with Rayleigh scattering in pure water.     

Magnetic nanoparticles as gene delivery agents: enhanced transfection in the presence of oscillating magnet arrays

Magnetic nanoparticle-based gene transfection has been shown to be effective in combination with both viral vectors and with non-viral agents. In these systems, therapeutic or reporter genes are attached to magnetic nanoparticles which are then focused to the target site/cells via high-field/high-gradient magnets. The technique has been shown to be efficient and rapid for in vitro transfection and compares well with cationic lipid-based reagents, producing good overall transfection levels with lower doses and shorter transfection times. In spite of its potential advantages (particularly for in vivo targeting), the overall transfection levels do not generally exceed those of other non-viral agents. In order to improve the overall transfection levels while maintaining the advantages inherent in this technique, we have developed a novel, oscillating magnet array system which adds lateral motion to the particle/gene complex in order to promote transfection. Experimental results indicate that the system significantly enhances overall in vitro transfection levels in human airway epithelial cells compared to both static field techniques (p<0.005) and the cationic lipids (p<0.001) tested. In addition, it has the previously demonstrated advantages of magnetofection-rapid transfection times and requiring lower levels of DNA than cationic lipid-based transfection agents. This method shows potential for non-viral gene delivery both in vitro and in vivo.     

Enhanced gene transfer with multilayered polyplexes assembled with layer-by-layer technique

Successful gene therapy asks for multifunctional vectors which can not only protect DNA from degradation but also transfer it into nuclear and subsequently express the loaded gene. Here we reported a novel multilayered delivery system constructed with DNA, protamine (Pro) and polyethylenimine (PEI) via lay-by-layer (LbL) technique, which posed multifunctions. DNA was previously condensed into a compact core with Pro which also contained nuclear localisation signals (NLS) domains for nuclear transfer. Then additional DNA was deposited as the first layer onto the cationic core via the electrostatic attraction which would increase the loading dose of DNA. At last, PEI was absorbed as the outmost layer to achieve the endosomal escape. Therefore a quaternary polyplexes which offered high loading of DNA, nuclear transfer ability and endosomal escape capability was constructed with the LbL technique. The obtained quaternary polyplexes showed positive surface charge, spherical morphology, a relatively narrow particle size distribution and strong DNA protection capability. Compared with commercially available PEI/DNA complexes, the novel multifuctional vector exhibited not only lower cytotoxicity (P<0.05) but also higher transfection efficiency in HepG2 and HeLa cells (P<0.05) in vitro test.     

Complete physicochemical characterization of DNA/chitosan complexes by multiple detection using asymmetrical flow field-flow fractionation

Asymmetrical flow field-flow fractionation (AF4) coupled with UV-vis spectrophotometry, multiangle light scattering (MALS), and dynamic light scattering (DLS) detection was used to analyze dispersions of DNA/rhodamine B labeled chitosan (Ch-rho) complexes frequently used as gene delivery vectors. The method yielded, in a single experiment, important characteristics of the complexes, such as their hydrodynamic radius, size distribution, conformation, composition, and the amount of free Ch-rho in the dispersions. Samples for analysis were obtained by varying experimental parameters known to influence the transfection efficiency of DNA/chitosan complexes, including the DNA concentration at mixing (82-164 μg/mL), the ratio of chitosan amino groups to DNA phosphate groups (3 ≤ N/P ratio ≤ 15), the chitosan molecular weight (10-76 kDa), and its degree of deacetylation. In all preparations, DNA/Ch-rho complexes had hydrodynamic radii ranging from 15 to 160 nm. Both the DNA concentration and the Ch-rho molecular weight influence the size distribution of the complexes: a greater fraction of large particles was detected in dispersions prepared with the most concentrated DNA solution or the Ch-rho of highest molar mass. All dispersions contained free Ch-rho in solution. The free Ch-rho content ranged from 53 to 92% of the total Ch-rho concentration in dispersions prepared with N/P ratios from 3 to 15, respectively, implying that the N/P ratio of the complexes ranged from 1.3 to 1.6 in all samples. The accuracy of the free Ch-rho determination by AF4/UV-vis/MALS+DLS was confirmed by an independent method involving (1) ultracentrifugation of the dispersions prepared with unlabeled chitosan and (2) analysis of the supernatant by the Orange II dye depletion method. This study demonstrates the ability of AF4/UV-vis/MALS+DLS to provide a complete physicochemical characterization of DNA/polycation complexes used in nonviral gene delivery.     

A data analysis algorithm for programmed field-flow fractionation

An algorithm that employs numerical integration for analysis of field-flow fractionation (FFF) data is presented. The algorithm utilizes detector response, field strength, and channel flow rate data, monitored at discrete time intervals during sample elution to generate a distribution of sample components according to particle size or molecular weight. The field strength and channel flow rate may either be held constant or programmed as functions of time, and it is not necessary for these programs to follow specific mathematical functions. If experimental conditions are monitored during a run, the algorithm can account for any deviation from nominal set conditions. The algorithm also allows calculation of fractionating power for the actual conditions as monitored during the run. The method provides greatly increased flexibility in the application of the FFF family of techniques. It removes the limitations on experimental conditions incurred by adherence to analytically available solutions to FFF theory, allowing ad hoc variation of field strength and other experimental parameters as necessary to increase sensitivity and specificity of the method. An implementation of the algorithm is described that is independent of the FFF technique (i.e., independent of field type) and mode of operation. To reduce computation time, it uses mathematical techniques to reduce the required number of numerical integrations. This is of particular importance when the perturbations to ideal FFF theory, such as those due to the effects of hydrodynamic lift forces, particle-wall or particle-particle interactions, and secondary relaxation, necessitate relatively lengthy numerical calculations.     

Novel drug delivery system by surface modified magnetic nanoparticles

In the recent progress of gene and cell therapy, novel drug delivery system (DDS) has been required for efficient delivery of small molecules/drugs and also the safety for clinical usage. We have already developed the unique transfection technique by preparing magnetic vector and using permanent magnet. This technique can improve the transfection efficiency. In this study, we directly associated plasmid DNA with magnetic nanoparticles, which can potentially enhance their transfection efficiency by magnetic force. Magnetic nanoparticle, such as magnetite, its average size of 18.7 nm, can be navigated by magnetic force and is basically consisted with oxidized Fe that is commonly used as the supplement drug for anemia. The magnetite particles coated with protamine sulfate, which gives a cationic surface charge onto the magnetite particle, significantly enhanced the transfection efficiency in vitro cell culture system. The magnetite particles coated with protamine sulfate also easily associated with cell surface, leading to high magnetic seeding percentage. From these results, it was found that the size and surface chemistry of magnetic particles would be tailored to meet specific demands on physical and biological characteristics accordingly. Overall, magnetic nanoparticles with different surface modification enhance the association with plasmid DNA and cell surface as well as HVJ-E, which potentially help to improve the drug delivery system.     

Charge shielding effects on gene delivery of polyethylenimine/DNA complexes: PEGylation and phospholipid coating

Polyethylenimine (PEI) is an efficient cationic polymer for gene delivery, but defective in biocompatibility. In this study, we developed two different strategies to shield the positively charged PEI/DNA complexes: PEGylation and lipid coating. The physicochemical properties, cytotoxicity and transfection efficiency of the two gene delivery systems were investigated. Both PEGylation and lipid coating succeeded in reducing the zeta-potential of the complexes. Lipid-coated PEI/DNA complexes (LPD complexes) and PEI/DNA complexes exhibited similar cytotoxicity, whereas PEG-PEI/DNA complexes showed lower cytotoxicity, especially at high N/P ratios. LPD complexes were less efficient in transfection compared to PEG-PEI/DNA complexes. The transfection efficiency was influenced remarkably by cytotoxicity and surface charge of the complexes. Intracellular processes studies revealed that endosomal release might be one of the rate-limiting steps in cell transfection with PEI as a gene delivery carrier.     

Protamine and chloroquine enhance gene delivery and expression mediated by RNA-wrapped single walled carbon nanotubes

The use of non-viral vectors as delivery systems in gene therapy has been extensively studied recently owing to their advantages over viral vectors. Here, we propose a new gene delivery system based on the use of RNA-wrapped single-walled carbon nanotubes (SWCNTs) complexed with the cationic protein, protamine and the drug chloroquine. Protamine was selected as a cationic protein acting as bridge between negatively charged RNA-wrapped SWCNTs and plasmid DNA. Protamine also contains a nuclear localization signal which enhances the expression of the transfected gene. The drug chloroquine, a lysosomotropic compound which has been reported to increase the transfection efficiency, was attached to RNA-wrapped SWNTs by ionic interactions. The simultaneous delivery of the drug chloroquine with plasmid DNA clearly showed an enhanced gene delivery and expression. The levels of gene expression were quantified using the luciferase reporter gene as model. Optimal conditions for transfection and gene expression were obtained and cytoxicity of the carbon nanotube complexes measured. The optimal complexes were shown to efficiently deliver plasmid DNA for efficient gene expression and may thereby be useful as gene delivery systems for gene therapy.     

Influence of the Number of Detectors for Laser Diffraction Scattering Method: Bimodal Distributions

The influence of the number of detectors is observed by comparing a given bimodal particle size distribution (PSD) and a calculated bimodal PSD by computer simulation and experimental method. A nonlinear iteration based on the Twomey method is used for calculating PSD. An observed range of scattering angles is 5.0 × 10^?4 to 2.5 rad, and the Mie light scattering theory is used. The number of detectors was changed from 6 to 81 elements by the computer simulations. The algorithm of the inversion problem is applied with mean diameters of lognormal distribution in a range from 0.546 to 422 µm at a standard deviation of 0.01 and 0.27. Experimental bimodal samples are prepared by certified monodisperse polystyrene latex standards, and results are obtained by three different kinds of optics, which has 21, 41, and 81 elements detector, respectively. The polydisperse bimodal PSD converges from 42 elements of the number of detectors. The bimodal PSD of monodisperses or size range of PSD under 50 µm converges from 21 elements of the number of detectors. The accuracy of calculated PSD is effected not only by the number of detectors but also the shape of the scattering function.     

Cationic liposome-DNA complexes: from liquid crystal science to gene delivery applications

At present, there is an unprecedented level of interest in the properties and structures of complexes consisting of DNA mixed with oppositely charged cationic liposomes (CLs). The interest arises because the complexes mimic natural viruses as chemical carriers of DNA into cells in worldwide human gene therapy clinical trials. However, since our understanding of the mechanisms of action of CL-DNA complexes interacting with cells remains poor, significant additional insights and discoveries will be required before the development of efficient chemical carriers suitable for long-term therapeutic applications. Recent studies describe synchrotron X-ray diffraction, which has revealed the liquid crystalline nature of CL-DNA complexes, and three-dimensional laser-scanning confocal microscopy, which reveals CL-DNA pathways and interactions with cells. The importance of the liquid crystalline structures in biological function is revealed in the application of these modern techniques in combination with functional transfection efficiency measurements, which shows that the mechanism of gene release from complexes in the cell cytoplasm is dependent on their precise liquid crystalline nature and the physical and chemical parameters (for example, the membrane charge density) of the complexes. In [section sign] 5, we describe some recent new results aimed at developing bionanotube vectors for gene delivery.