Kelvin probe force microscopy to study electrostatic interactions of DNA with lipid-gemini surfactant monolayers for gene delivery.

In novel gene therapy mechanisms utilising gemini surfactants, electrostatic interactions of the surfactant molecules with the DNA strands is a primary mechanism by which the two components of the delivery vehicle bind. In this work, we show for the first time direct evidence of electrostatic interactions of these compounds visualised with Kelvin probe force microscopy (KPFM) and correlated to their topography from atomic force microscopy (AFM). We construct monolayers of lipids and gemini surfactant to simulate interactions on a cellular level, using lipids commonly found in cell membranes, and allow DNA to bind to the monolayer as it is formed on a Langmuir-Blodgett trough. The difference in topography and electrical surface potential between monolayers with and without DNA is striking. In fact, KPFM reveals a strongly positive relative electrical surface potential in between where we identify a background lipid and the DNA strands, evidenced by the height profiles of the domains. Such identification is not possible without KPFM. We conclude that it is likely we are seeing cationic surfactant molecules surrounding DNA strands within a sea of background lipid.

Continuous Langmuir-Blodgett Deposition and Transfer by Controlled Edge-to-Edge Assembly of Floating 2D Materials.

The Langmuir-Blodgett technique is one of the most controlled methods to deposit monomolecular layers of floating or surface active materials but has lacked the ability to coat truly large-area substrates. In this work, by manipulating single-layer dispersions of graphene oxide (GO) and thermally exfoliated GO into water-immiscible spreading solvents, unlike traditional Langmuir-Blodgett deposition which requires densification achieved by compressing barriers, we demonstrate the ability to control the 2D aggregation and densification behavior of these floating materials using a barrier-free method. This is done by controlling the edge-to-edge interactions through modified subphase conditions and by utilizing the distance-dependent spreading pressure of the deposition solvent. These phenomena allow substrates to be coated by continuous deposition and substrate withdrawal-enabling roll-to-roll deposition and patterning of large-area substrates such as flexible polyethylene terephthalate. The aggregation and solvent-driven densification phenomena are examined by in situ Brewster angle video microscopy and by measuring the local spreading pressure induced by the spreading solvent acting on the floating materials using a Langmuir-Adam balance. As an example, the performance of films deposited in this way is assessed as passivation layers for Ag nanowire-based transparent conductors.

Synthesis and characterization of asymmetrical gemini surfactants.

The effect of variation in the length of surfactant hydrocarbon tail groups was tested in a series of dissymmetric gemini surfactants (N1-alkyl N1,N1,N3,N3-tetramethyl-N3-(6-pyren-6yl)-hexyl)propane-1,3-diammonium dibromide designated as CmC3CnBr, with m = hexyl pyrene, and n = 8, 12, 14, 16, and 18. The aggregation properties of these surfactants have been investigated by means of 1H NMR, fluorescence spectroscopy, surface tension and electrical conductivity measurements. The critical micelle concentration (CMC) was determined using surface tension and confirmed using the specific conductance method. Krafft temperatures and the degree of micelle ionization (α) were obtained from specific conductance measurements. With an increase of the dissymmetry (m/n) ratio, the CMC decreased linearly and an increase in the Krafft temperatures was observed for all of the gemini surfactants. α values for the dissymmetric GS were higher than those of the m-3-m counterparts, which may be attributed to enhanced micelle-micelle interactions that arise from increased hydrophobicity of the hydrocarbon chains. The introduction of the bulky pyrenyl tail group resulted in much lower CMC values compared to their symmetrical counterparts affecting the packing of these surfactants at the air/water interface, which resulted in high-ordered structures (lamellar and inverted micelles). This in turn affected the thermodynamic parameters of the micellization.

Synergistic behaviour of ZnO nanoparticles and gemini surfactants on the dynamic and equilibrium oil/water interfacial tension.

In this work the effect of ZnO nanoparticles on the interfacial behaviour of gemini surfactants (12-3-12 and 14-3-14) at the oil/water interface was investigated. Equilibrium and dynamic interfacial tension in the absence and presence of ZnO was measured and compared. The results show that the synergistic interactions between the surfactants and nanoparticles decrease the interfacial tension beyond that observed for each component, alone. Modelling of dynamic data with two different models indicates that the mechanism of surfactant migration (with and without ZnO) is mixed diffusion-kinetic-control. The Gibbs free energy of micellization and the Gibbs free energy of adsorption in the absence and presence of ZnO were calculated and compared. Finally the effect of addition of ZnO nanoparticles on emulsion stability was also examined.

Nanoscale Structure of Lipid-Gemini Surfactant Mixed Monolayers Resolved with AFM and KPFM Microscopy.

Drug delivery vehicles composed of lipids and gemini surfactants (GS) are promising in gene therapy. Tuning the composition and properties of the delivery vehicle is important for the efficient load and delivery of DNA fragments (genes). In this paper, we studied novel gene delivery systems composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-3-phosphocholine (DPPC), and GS of the type N,N-bis(dimethylalkyl)-α,ω-alkanediammonium dibromide at different ratios. The nanoscale properties of the mixed DOPC-DPPC-GS monolayers on the surface of the gene delivery system were studied using atomic force microscopy (AFM) and Kelvin probe force microscopy (KPFM). We demonstrate that lipid-GS mixed monolayers result in the formation of nanoscale domains that vary in size, height, and electrical surface potential. We show that the presence of GS can impart significant changes to the domain topography and electrical surface potential compared to monolayers composed of lipids alone.

Effect of chemical permeation enhancers on stratum corneum barrier lipid organizational structure and interferon alpha permeability.

The outermost layer of the skin, known as the stratum corneum (SC), is composed of dead corneocytes embedded in an intercellular lipid matrix consisting of ceramides, free fatty acids, and cholesterol. The high level of organization within this matrix protects the body by limiting the permeation of most compounds through the skin. While essential for its protective functions, the SC poses a significant barrier for the delivery of topically applied pharmaceutical agents. Chemical permeation enhancers (CPEs) can increase delivery of small drug compounds into the skin by interacting with the intercellular lipids through physical processes including extraction, fluidization, increased disorder, and phase separation. However, it is not clear whether these same mechanisms are involved in delivery of biotherapeutic macromolecules, such as proteins. Here we describe the effect of three categories of CPEs {solvents [ethanol, propylene glycol, diethylene glycol monoethyl ether (transcutol), oleic acid], terpenes [menthol, nerol, camphor, methyl salicylate], and surfactants [Tween 80, SDS, benzalkonium chloride, polyoxyl 40 hydrogenated castor oil (Cremophor RH40), didecyldimethylammonium bromide (DDAB), didecyltrimethylammonium bromide (DTAB)]} on the lipid organizational structure of human SC as determined by X-ray scattering studies. Small- and wide-angle X-ray scattering studies were conducted to correlate the degree of structural changes and hydrocarbon chain packing in SC lipids caused by these various classes of CPEs to the extent of permeation of interferon alpha-2b (IFNα), a 19 kDa protein drug, into human skin. With the exception of solvents, propylene glycol and ethanol, all classes of CPEs caused increased disordering of lamellar and lateral packing of lipids. We observed that the highest degree of SC lipid disordering was caused by surfactants (especially SDS, DDAB, and DTAB) followed by terpenes, such as nerol. Interestingly, in vitro skin permeation studies indicated that, in most cases, absorption of IFNα was low and that an increase in SC lipid disorder does not correspond to an increase in IFNα absorption.

Transfection and structural properties of phytanyl substituted gemini surfactant-based vectors for gene delivery.

In this study, the transfection ability and cytotoxicity of a series of phytanyl substituted gemini surfactants, rationally designed and synthesized in an attempt to create cationic surfactants that will improve transfection efficiencies of non-viral vectors was evaluated in OVCAR-3 cells at the charge ratios (N(+)/P(-)) of 2:1, 5:1, and 10:1. Particle sizes, zeta potentials, and small angle X-ray scattering (SAXS) profiles were also determined. For each gemini surfactant complex, the transfection efficiency and cytotoxicity are observed to go through a more or less well-evidenced maximum, occurring at different values of the charge ratio (N(+)/P(-)), depending on the surfactant structure. Considering both results of in vitro transfection efficiency and cytotoxicity, the optimal charge ratio to formulate the complexes containing phy-3-m was found to be 5:1. The particle size decreased, while zeta potential increased with increasing N(+)/P(-). Comparing particle size and zeta potential with transfection efficiency, no correlation between size/zeta potential and transfection ability was observed. Analysis of SAXS profiles indicates that the ability of phy-3-m delivery system to adopt multiple phases correlated well with their higher transfection efficiency in OVCAR-3 cells.

Defect-Rich MoSe2 2H/1T Hybrid Nanoparticles Prepared from Femtosecond Laser Ablation in Liquid and Their Enhanced Photothermal Conversion Efficiencies.

MoSe2 2H/1T hybrid nanoparticles are prepared by femtosecond laser ablation of MoSe2 powder in isopropyl alcohol with different laser powers and ablation times, and their formation mechanisms and photothermal conversion efficiencies (PTCEs) are studied. Two types of spherical nanoparticles are observed. The first type is onion-structured nanoparticles that are formed by nucleation on the surfaces of melted droplets followed by inward growth of {002} planes of MoSe2 . The second type is polycrystalline nanoparticles, formed by coalescence of crystalline nanoclusters fragmented from the powder during the laser ablation. The nanoparticle size in all samples shows a bimodal distribution, corresponding to different fragmentation mechanisms. The 2H-to-1T phase transition in the nanoparticles is likely caused by electron doping from the laser-induced plasma. The PTCEs of the nanoparticles increase with laser power and ablation time; the highest PTCE is around 38%. After examining the bandgaps and the Urbach energies of the nanoparticles, it is found that the high PTCEs are primarily attributed to defects and structural disorder in the laser-synthesized nanoparticles, which allow absorption of photons with energies smaller than the bandgap energy and facilitate non-radiative recombination of photoexcited carriers.

Aryl hydrocarbon receptor nuclear translocator/hypoxia-inducible factor-1{beta} plays a critical role in maintaining glucose-stimulated anaplerosis and insulin release from pancreatic {beta}-cells.

The metabolic pathways that are involved in regulating insulin secretion from pancreatic ß-cells are still incompletely understood. One potential regulator of the metabolic phenotype of ß-cells is the transcription factor aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia-inducible factor (HIF)-1ß. ARNT/HIF-1ß levels are profoundly reduced in islets obtained from type 2 diabetic patients. However, no study to date has investigated key pathways involved in regulating insulin release in ß-cells that lack ARNT/HIF-1ß. In this study, we confirm that siRNA-mediated knockdown of ARNT/HIF-1ß inhibits glucose-stimulated insulin secretion. We next investigated the metabolic consequence of the loss of ARNT/HIF-1ß knockdown. We demonstrate that ß-cells with reduced ARNT/HIF-1ß expression levels exhibit a 31% reduction in glycolytic flux without significant changes in glucose oxidation or the ATP:ADP ratio. Metabolic profiling of ß-cells treated with siRNAs against the ARNT/HIF-1ß gene revealed that glycolysis, anaplerosis, and glucose-induced fatty acid production were down-regulated, and all are key events involved in glucose-stimulated insulin secretion. In addition, both first and second phase insulin secretion in islets were significantly reduced after ARNT/HIF-1ß knockdown. Together, our data suggest an important role for ARNT/HIF-1ß in anaplerosis, and it may play a critical role in maintaining normal secretion competence of ß-cells.

An Overview of Nanotechnologies for Drug Delivery to the Brain.

Drug delivery to the brain has been one of the toughest challenges researchers have faced to develop effective treatments for brain diseases. Owing to the blood-brain barrier (BBB), only a small portion of administered drug can reach the brain. A consequence of that is the need to administer a higher dose of the drug, which, expectedly, leads to a variety of unwanted side effects. Research in a variety of different fields has been underway for the past couple of decades to address this very serious and frequently lethal problem. One area of research that has produced optimistic results in recent years is nanomedicine. Nanomedicine is the science birthed by fusing the fields of nanotechnology, chemistry and medicine into one. Many different types of nanomedicine-based drug-delivery systems are currently being studied for the sole purpose of improved drug delivery to the brain. This review puts together and briefly summarizes some of the major breakthroughs in this crusade. Inorganic nanoparticle-based drug-delivery systems, such as gold nanoparticles and magnetic nanoparticles, are discussed, as well as some organic nanoparticulate systems. Amongst the organic drug-delivery nanosystems, polymeric micelles and dendrimers are discussed briefly and solid polymeric nanoparticles are explored in detail.

Characterization of the behavior of a pyrene substituted gemini surfactant in water by fluorescence.

Time-resolved fluorescence was applied to characterize the behavior in solution of a gemini surfactant substituted with pyrene (Py-3-12). Upon association in water, excimer formation by Py-3-12 can be probed by acquiring pyrene monomer and excimer fluorescence decays which can be fitted globally according to the model free (MF) analysis to yield quantitative information about the internal dynamics of the Py-3-12 surfactant micelles as well as a complete description of the distribution of the different pyrene species in solution either incorporated inside the micelles or free in solution. A proof of procedure for the MF analysis was established by noting that the concentrations of free surfactant in solution, [Py-3-12](free), was found to equal the critical micelle concentration (CMC) for surfactant concentrations larger than the CMC. (I(E)/I(M))(SPC), the ratio of pyrene monomer to excimer fluorescence intensities, was calculated from parameters retrieved from the MF analysis of the fluorescence decays and was found to be independent of sample geometry. This work demonstrates how time-resolved fluorescence can be used to study the properties of pyrene-labeled macromolecules under conditions where large absorptions and inner filter effects usually distort the steady-state fluorescence signals. It was found that the pyrene excimer is formed mostly by diffusion within the Py-3-12 micelles, which suggests that the pyrene microenvironment is fluid, an important feature for future studies on the interactions of Py-3-12 with DNA.

Synthesis and aggregation properties of dissymmetric phytanyl-gemini surfactants for use as improved DNA transfection vectors.

Improvements in transfection efficiency are required in order to make the goal of cellular gene delivery by non-viral vectors realizable. Novel derivatives of gemini surfactants having dissymmetric tail groups have been designed specifically as a means to improve DNA transfection; the micelle and interfacial properties are reported herein. The effect of these substitutions on the aggregation properties of the gemini surfactants is discussed in the context of results for the m-3-m gemini series, previously reported in the literature. Phytanyl substitution results in lower cmc and higher micelle ionization. In addition, the phytanyl substituted gemini surfactants form vesicles at room temperature. Preliminary in vitro transfection assays showed the phytanyl substituted gemini surfactants to be more efficient transfection vectors as compared to symmetric gemini surfactants.

Challenges of Dissolution Methods Development for Soft Gelatin Capsules.

Recently, the development of soft gelatin capsules (SGCs) dosage forms has attracted a great deal of interest in the oral delivery of poorly water-soluble drugs. This is attributed to the increased number of poorly soluble drugs in the pipeline, and hence the challenges of finding innovative ways of developing bioavailable and stable dosage forms. Encapsulation of these drugs into SGCs is one of the approaches that is utilized to deliver the active ingredients to the systemic circulation to overcome certain formulation hurdles. Once formulated, encapsulated drugs in the form of SGCs require suitable in vitro dissolution test methods to ensure drug product quality and performance. This review focuses on challenges facing dissolution test method development for SGCs. A brief discussion of the physicochemical and formulation factors that affect the dissolution properties of SGCs will be highlighted. Likewise, the influence of cross-linking of gelatin on the dissolution properties of SGCs will also be discussed.

Topical delivery of interferon alpha by biphasic vesicles: evidence for a novel nanopathway across the stratum corneum.

Noninvasive delivery of macromolecules across intact skin is challenging but would allow for needle-free administration of many pharmaceuticals. Biphasic vesicles, a novel lipid-based topical delivery system, have been shown to deliver macromolecules into the skin. Investigation of the delivery mechanism of interferon alpha (IFN alpha), as a model protein, by biphasic vesicles could improve understanding of molecular transport through the stratum corneum and allow for the design of more effective delivery systems. The interaction of biphasic vesicles with human skin and isolated stratum corneum membrane was investigated by confocal microscopy, differential scanning calorimetry (DSC) and small- and wide-angle X-ray scattering (SAXS and WAXS). Confocal microscopy revealed that biphasic vesicles delivered IFN alpha intercellularly, to a depth of 70 microm, well below the stratum corneum and into the viable epidermis. DSC and SAXS/WAXS data suggest that the interaction of biphasic vesicles with SC lipids resulted in the formation of a three-dimensional cubic Pn3m polymorphic phase by the molecular rearrangement of intercellular lipids. This cubic phase could be an intercellular permeation nanopathway that may explain the increased delivery of IFN alpha by biphasic vesicles. Liposomes and submicrometer emulsion (the individual building blocks of biphasic vesicles) separately and methylcellulose gel, an alternative topical vehicle, did not induce a cubic phase and delivered low amounts of IFN alpha below the stratum corneum. Molecular modeling of the cubic Pn3m phase and lamellar-to-cubic phase transitions provides a plausible mechanism for transport of IFN alpha. It is hypothesized that induction of a Pn3m cubic phase in stratum corneum lipids could make dermal and transdermal delivery of other macromolecules also possible.

Thermodynamic investigation of the binding of dissymmetric pyrenyl-gemini surfactants to DNA.

Gemini surfactants have demonstrated significant potential for use in constructing non-viral transfection vectors for the delivery of genes into cells to induce protein expression. Previously, two asymmetric gemini surfactants containing pyrenyl groups in one of the alkyl tails of the surfactants were synthesized as fluorescence probes for use in mechanistic studies of the transfection process. Here we present the results of a thermodynamic investigation of the binding interaction(s) between the pyrenyl-modified surfactants and DNA. The thermodynamics of the interactions have been examined using isothermal titration calorimetry, light scattering, zeta potential, and circular dichroism measurements. Distinct differences are observed between the interaction of 12-s-12 vs. the pyrene modified py-s-12 surfactants with DNA; an intercalated binding is found for the py-s-12 surfactants that disrupts the typical interactions observed between DNA and gemini surfactants.

m-s-m cationic gemini and zwitterionic surfactants - a thermodynamic analysis of their mixed micelle formation.

Micelle formation enthalpies (Δmic H values) have been calorimetrically determined at 298 K for three sets of mixed zwitterionic/cationic gemini systems consisting of N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (ZW3-12) and a series of structurally related gemini surfactants, the N,N'-bis(dimethyldodecyl)-α,ω-alkanediammonium dibromide (12-s-12) systems. From the experimental and the estimated ideal micelle formation enthalpies, the excess enthalpies were obtained. The degrees of nonideality of the interaction in the mixed micelle (ß m) from our previous work was used along with the excess enthalpy values to determine excess thermodynamic quantities of the surfactants in the mixed system according to Regular Solution Theory (RST) and Motomura's theory. The excess enthalpies for the ZW3-12/12-4-12 were positive in magnitude and rose sharply when small amounts of the zwittergent were distributed into the gemini micelles. The excess enthalpies for the ZW3-12/12-5-12 and the ZW3-12/12-6-12 systems were also >0 kJ mol-1, and as a function of zwittergent composition, were quite different to those of the ZW3-12/12-4-12 mixed micelles. These results indicate that the heat of mixed micelle formation is strongly dependent on electrostatic interactions and the structure of the surfactants involved, specifically, the length of the tether group for the 12-s-12 gemini surfactants. From the calorimetric data and the application of RST and Motomura's theory, we have obtained estimates of the excess Gibbs energy and entropy of mixing. An analysis of the three thermodynamic properties suggests that the relative contributions of enthalpic and entropic effects to nonideal behavior for mixed micelles involving gemini surfactants are strongly dependent on the gemini structure.

Avocado-derived polyols for use as novel co-surfactants in low energy self-emulsifying microemulsions.

Avocado (Persea americana Mill.; Lauraceae) seed-derived polyhydroxylated fatty alcohols (PFAs) or polyols (i.e., avocadene and avocadyne) are metabolic modulators that selectively induce apoptosis of leukemia stem cells and reverse pathologies associated with diet-induced obesity. Delivery systems containing avocado polyols have not been described. Herein, natural surface active properties of these polyols are characterized and incorporated into self-emulsifying drug delivery systems (SEDDS) that rely on molecular self-assembly to form fine, transparent, oil-in-water (O/W) microemulsions as small as 20 nanometers in diameter. Mechanistically, a 1:1 molar ratio of avocadene and avocadyne (i.e., avocatin B or AVO was shown to be a eutectic mixture which can be employed as a novel, bioactive, co-surfactant that significantly reduces droplet size of medium-chain triglyceride O/W emulsions stabilized with polysorbate 80. In vitro cytotoxicity of avocado polyol-SEDDS in acute myeloid leukemia cell lines indicated significant increases in potency and bioactivity compared to conventional cell culture delivery systems. A pilot pharmacokinetic evaluation of AVO SEDDS in C57BL/6J mice revealed appreciable accumulation in whole blood and biodistribution in key target tissues. Lastly, incorporation of AVO in SEDDS significantly improved encapsulation of the poorly water-soluble drugs naproxen and curcumin.

Modified gelatin nanoparticles for gene delivery.

Gelatin nanoparticles (GNPs) are one of the most extensively used natural polymers for gene therapy. With advantages of being biodegradable, biocompatible, low cost and easily modified, gelatin holds great promise as a non-viral system for gene delivery. This review examines various methods of preparation of modified gelatin nanoparticles and considers how these modifications apply to gene delivery. The article discusses cationic gelatin, PEGylated gelatin, thiolated gelatin, alendronate gelatin, and EGFR gelatin nanoparticles. This article also considers several advantages of these modifications and their contribution to the improvement in the efficiency of these systems, resulting in superior transfection and enhanced gene delivery in general.

Investigating the Phospholipid Effect on the Bioaccessibility of Rosmarinic Acid-Phospholipid Complex through a Dynamic Gastrointestinal in Vitro Model.

Phyto-phospholipid complexes have been developed as a common way of improving the oral bioavailability of poorly absorbable phyto-pharmaceuticals; however, the complexation with phospholipids can induce positive or negative effects on the bioaccessibility of such plant-derived active ingredients in different parts of the gastrointestinal tract (GIT). The purpose of this study was to investigate the effects of phospholipid complexation on the bioaccessibility of a rosmarinic acid-phospholipid complex (RA-PLC) using the TNO dynamic intestinal model-1 (TIM-1). Preparation of RA-PLC was confirmed using X-ray diffraction, Fourier-transform infrared spectroscopy, partition coefficient measurement, and Caco-2 monolayer permeation test. Bioaccessibility parameters in different GIT compartments were investigated. Complexation by phospholipids reduced the bioaccessibility of RA in jejunum compartment, while maintaining the ileum bioaccessibility. The overall bioaccessibility of RA-PLC was lower than the unformulated drug, suggesting that the improved oral absorption from a previous animal study could be considered as a net result of decreased bioaccessibility overwhelmed by enhanced intestinal permeability. This study provides insights into the effects of phospholipid on the bioaccessibility of hydrophilic compounds, and analyzes them based on the relationship between bioaccessibility, membrane permeability, and bioavailability. Additionally, TIM-1 shows promise in the evaluation of dosage forms containing materials with complicated effects on bioaccessibility.

Biodistribution and Physiologically-Based Pharmacokinetic Modeling of Gold Nanoparticles in Mice with Interspecies Extrapolation.

Gold nanoparticles (AuNPs) are a focus of growing medical research applications due to their unique chemical, electrical and optical properties. Because of uncertain toxicity, "green" synthesis methods are emerging, using plant extracts to improve biological and environmental compatibility. Here we explore the biodistribution of green AuNPs in mice and prepare a physiologically-based pharmacokinetic (PBPK) model to guide interspecies extrapolation. Monodisperse AuNPs were synthesized and capped with epigallocatechin gallate (EGCG) and curcumin. 64 CD-1 mice received the AuNPs by intraperitoneal injection. To assess biodistribution, groups of six mice were sacrificed at 1, 7, 14, 28 and 56 days, and their organs were analyzed for gold content using inductively coupled plasma mass spectrometry (ICP-MS). A physiologically-based pharmacokinetic (PBPK) model was developed to describe the biodistribution data in mice. To assess the potential for interspecies extrapolation, organism-specific parameters in the model were adapted to represent rats, and the rat PBPK model was subsequently evaluated with PK data for citrate-capped AuNPs from literature. The liver and spleen displayed strong uptake, and the PBPK model suggested that extravasation and phagocytosis were key drivers. Organ predictions following interspecies extrapolation were successful for rats receiving citrate-capped AuNPs. This work lays the foundation for the pre-clinical extrapolation of the pharmacokinetics of AuNPs from mice to larger species.