Synthesis of biodegradable polymer-mesoporous silica composite microspheres for DNA prime-protein boost vaccination

DNA vaccines or proteins are capable of inducing specific immunity; however, the translation to the clinic has generally been problematic, primarily due to the reduced magnitude of immune response and poor pharmacokinetics. Herein we demonstrate a composite microsphere formulation, composed of mesoporous silica spheres (MPS) and poly(d,l-lactide-co-glycolide) (PLGA), enables the controlled delivery of a prime-boost vaccine via the encapsulation of plasmid DNA (pDNA) and protein in different compartments. Method with modified dual-concentric-feeding needles attached to a 40 kHz ultrasonic atomizer was studied. These needles focus the flow of two different solutions, which passed through the ultrasonic atomizer. The process synthesis parameters, which are important to the scale-up of composite microspheres, were also studied. These parameters include polymer concentration, feed flowrate, and volumetric ratio of polymer and pDNA-PEI/MPS-BSA. This fabrication technique produced composite microspheres with mean D[4,3] ranging from 6 to 34 μm, depending upon the microsphere preparation. The resultant physical morphology of composite microspheres was largely influenced by the volumetric ratio of pDNA-PEI/MPS-BSA to polymer, and this was due to the precipitation of MPS at the surface of the microspheres. The encapsulation efficiencies were predominantly in the range of 93-98% for pDNA and 46-68% for MPS. In the in vitro studies, the pDNA and protein showed different release kinetics in a 40 day time frame. The dual-concentric-feeding in ultrasonic atomization was shown to have excellent reproducibility. It was concluded that this fabrication technique is an effective method to prepare formulations containing a heterologous prime-boost vaccine in a single delivery system.

Preparation and characterization of poly(lactic-co-glycolic acid) microparticles containing DNA molecules encoding a malaria vaccine candidate

Background A novel ultrasonic atomization approach for the formulation of biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles of a malaria DNA vaccine is presented. A 40 kHz ultrasonic atomization device was used to create the microparticles from a feedstock containing 5 volumes of 0.5% w/v PLGA in acetone and 1 volume of condensed DNA which was fed at a flow rate of 18ml h-1. The plasmid DNA vectors encoding a malaria protein were condensed with a cationic polymer before atomization. Results High levels of gene expression in vitro were observed in COS-7 cells transfected with condensed DNA at a nitrogen to phosphate (N/P) ratio of 10. At this N/P ratio, the condensed DNA exhibited a monodispersed nanoparticle size (Z-average diameter of 60.8 nm) and a highly positive zeta potential of 38.8mV. The microparticle formulations of malaria DNA vaccine were quality assessed and it was shown that themicroparticles displayed high encapsulation efficiencies between 82-96% and a narrow size distribution in the range of 0.8-1.9 μm. In vitro release profile revealed that approximately 82% of the DNA was released within 30 days via a predominantly diffusion controlledmass transfer system. Conclusions This ultrasonic atomization technique showed excellent particle size reproducibility and displayed potential as an industrially viable approach for the formulation of controlled release particles.

Melt electrospinning of poly(epsilon-caprolactone) scaffolds: Phenomenological observations associated with collection and direct writing

Melt electrospinning and its additive manufacturing analogue, melt electrospinning writing (MEW), are two processes which can produce porous materials for applications where solvent toxicity and accumulation in solution electrospinning are problematic. This study explores the melt electrospinning of poly(ε-caprolactone) (PCL) scaffolds, specifically for applications in tissue engineering. The research described here aims to inform researchers interested in melt electrospinning about technical aspects of the process. This includes rapid fiber characterization using glass microscope slides, allowing influential processing parameters on fiber morphology to be assessed, as well as observed fiber collection phenomena on different collector substrates. The distribution and alignment of melt electrospun PCL fibers can be controlled to a certain degree using patterned collectors to create large numbers of scaffolds with shaped macroporous architectures. However, the buildup of residual charge in the collected fibers limits the achievable thickness of the porous template through such scaffolds. One challenge identified for MEW is the ability to control charge buildup so that fibers can be placed accurately in close proximity, and in many centimeter heights. The scale and size of scaffolds produced using MEW, however, indicate that this emerging process will fill a technological niche in biofabrication.

Electric Field–Enhanced Sensitivity of Grafted Ligands and Receptors

BACKGROUND: Particle-based agglutination tests consisting of receptors grafted to colloidal microparticles are useful for detecting small quantities of corresponding ligands of interest in fluid test samples, but detection limits of such tests are limited to a certain concentration and it is most desirable to lower the detection limits and to enhance the rate of recognition of ligands. METHODS: A mixture of receptor-coated colloidal microparticles and corresponding ligand was sandwiched between 2 indium tin oxide-coated glass plates. Electrohydrodynamic drag from an alternating-current electric field applied perpendicular to the plates increased the local concentration of the colloidal particles, improving the chances of ligand-receptor interaction and leading to the aggregation of the colloidal particles. RESULTS: With this technique the sensitivity of the ligand-receptor recognition was increased by a factor as large as 50. CONCLUSIONS: This method can improve the sensitivity of particle-based agglutination tests used in immuno-assays and many other applications such as immunoprecipitation and chemical, sniffing. (C) 2007 American Association for Clinical Chemistry.

New type of high field electrooptic response in nematics

We have made concurrent measurements of ionic current and optical transmission between crossed polarisers on several nematics with positive dielectric anisotropy under the action of applied low frequency (< 1KHz) square wave voltages. When the field E is low, the measured current is linear in E and there is no electrooptic response. Beyond some value of the field (E(0)similar to 100 esu), the current becomes independent of the field (phenomenon of limiting current). Further an electrooptic signal is measured at twice the frequency of the applied voltage, which exhibits a peak as a function of the field. The width of the peak is 3 to 4 times the value of E-0, and the signal level at the peak decreases as the frequency is increased. These measurements have been made on three highly polar compounds with cyano end groups. Careful observations do not show any evidence of electrohydrodynamic instabilities in the sample. It is argued that the observations can be understood if at the onset of the phenomenon of the limiting current, a strong electric field gradient is established near one of the electrodes due to the sweeping of an ionic species with high mobility. The field gradient produces a flexoelectric deformation of the director field, which in turn gives rise to the electrooptic effect. At higher fields, the stabilising dielectric torque takes over to suppress this instability.

Metastable extension of the fluorite phase field in Y2O3---ZrO2 and its effect on grain growth

Pure Y2O3 and Y2O3---ZrO2 solid solutions have been prepared by melt atomization and by pyrolysis of nitrate solutions. Extended solubility is readily achieved in both techniques for the entire composition range investigated: melts with 0–30% ZrO2 and precursors with 0–50% ZrO2. However, solidification of under cooled droplets yields almost exclusively single phase powders with the structure of cubic yttria (D53). In contrast, the pyrolysis route leads to a sequence of metastable microstructures beginning with a nanocrystalline disordered fluorite-based (C1) solid solution. Further heating leads to the evolution of much larger (micron size) flake crystals with a {001} texture, concurrent with partial ordering of the oxygen ions to the sites occupied in the D53 structure. The driving force for ordering and the rate of grain growth decrease with increasing ZrO2 addition. Abrupt heating to high temperatures or electron irradiation can induce ordering without substantial grain growth. There is no significant reduction in porosity during the recrystallization, which with the other observations suggests that grain growth is driven by the free energy available for the ordering transformation from fluorite to the yttria structure. This route offers opportunities for single crystal thin film development at relatively low processing temperatures.

Metastable extension of the fluorite phase field in Y2O3---ZrO2 and its effect on grain growth

Pure Y2O3 and Y2O3-ZrO2 solid solutions have been prepared by melt atomization and by pyrolysis of nitrate solutions. Extended solubility is readily achieved in both techniques for the entire composition range investigated: melts with 0-30% ZrO2 and precursors with 0-50% ZrO2. However, solidification of under cooled droplets yields almost exclusively single phase powders with the structure of cubic yttria (D5(3)). In contrast, the pyrolysis route leads to a sequence of metastable microstructures beginning with a nanocrystalline disordered fluorite-based (C1) solid solution. Further heating leads to the evolution of much larger (micron size) flake crystals with a {001} texture, concurrent with partial ordering of the oxygen ions to the sites occupied in the D5(3) structure. The driving force for ordering and the rate of grain growth decrease with increasing ZrO2 addition. Abrupt heating to high temperatures or electron irradiation can induce ordering without substantial grain growth. There is no significant reduction in porosity during the recrystallization, which with the other observations suggests that grain growth is driven by the free energy available for the ordering transformation from fluorite to the yttria structure. This route offers opportunities for single crystal thin film development at relatively low processing temperatures.

Dispersion and vaporization of biofuels and conventional fuels in a crossflow pre-mixer

In lean premixed pre-vaporized (LPP) combustion, controlled atomization, dispersion and vaporization of different types of liquid fuel in the premixer are the key factors required to stabilize the combustion process and improve the efficiency. The dispersion and vaporization process for biofuels and conventional fuels sprayed into a crossflow pre-mixer have been simulated and analyzed with respect to vaporization rate, degree of mixedness and homogeneity. Two major biofuels under investigation are Ethanol and Rapeseed Methyl Esters (RME), while conventional fuels are gasoline and jet-A. First, the numerical code is validated by comparing with the experimental data of single n-heptane and decane droplet evaporating under both moderate and high temperature convective air now. Next, the spray simulations were conducted with monodispersed droplets with an initial diameter of 80 mu m injected into a turbulent crossflow of air with a typical velocity of 10 m/s and temperature of around 800K. Vaporization time scales of different fuels are found to be very different. The droplet diameter reduction and surface temperature rise were found to be strongly dependent on the fuel properties. Gasoline droplet exhibited a much faster vaporization due a combination of higher vapor pressure and smaller latent heat of vaporization compared to other fuels. Mono-dispersed spray was adopted with the expectation of achieving more homogeneous fuel droplet size than poly-dispersed spray. However, the diameter histogram in the zone near the pre-mixer exit shows a large range of droplet diameter distributions for all the fuels. In order to improve the vaporization performance, fuels were pre-heated before injection. Results show that the Sauter mean diameter of ethanol improved from 52.8% of the initial injection size to 48.2%, while jet-A improved from 48.4% to 18.6% and RME improved from 63.5% to 31.3%. The diameter histogram showed improved vaporization performance of jet-A. (C) 2011 Elsevier Ltd. All rights reserved.

Mass Distribution Studies in Effervescent Sprays

An experimental study on liquid mass distribution in effervescent sprays using water and air as working fluids is presented in this paper. Optical patternation and techniques of image processing are employed for analyzing the spray. The flow regime inside the effervescent atomizer largely dictates the mass distribution patterns. The patterns are seen to vary from concentrated, poorly atomized liquid lumps to uniformly distributed, fine droplets as the flow regime changes from bubbly flow to annular flow. A large variety of instantaneous spray patterns are observed in bubbly flow regime indicating a highly unsteady atomization process. However, relatively better consistency in spray patterns is observed at higher gas flow rates. Thus, the degree of unsteadiness gradually diminishes as gas flow rate is increased. The axial evolution of the spray in annular mode shows good mixing of liquid and gas across the interface.

Experimental Studies on High-Pressure Spray Structure of Biofuels

The high-pressure spray characteristics of biofuels, specifically, Pongamia oil and its blends with diesel are studied for various gas pressures. Two single-hole solenoid injectors with nozzle diameters of 200 and 260 mu m are used along with a high-pressure common-rail direct-injection system to inject fuel into a high-pressure spray visualization chamber. The spray structure is characterized using a high-speed laser-based shadowgraphy technique. The spray structure of Pongamia oil revealed the presence of an intact liquid core at low gas pressure. At high gas pressures, the spray atomization of the Pongamia oil showed marked improvement. The spray tip penetration of Pongamia oil and its blends with diesel is higher compared to that of diesel for all test conditions. The spray cone angle of Pongamia oil and 50% Pongamia oil blend with diesel is lower as compared to that of diesel. Both these observations are attributed to the presence of large droplets carrying higher momentum in oil and blend. The droplet size is measured at an injection pressure of 1000 bar and gas pressure of 30 bar at 25 mm below the nozzle tip using the particle/droplet image.analysis (PDIA) method. The droplet size measurements have shown that the Sauter mean diameter (SMD) in the spray core of Pongamia oil is more than twice that of diesel. The spray tip penetration of the 20% blend of Pongamia with diesel (P20) is similar to that of diesel but the SMD is 50% higher. Based on experimental data, appropriate spray tip penetration correlation is proposed for the vegetable oil fuels such as Pongamia.

Spray characterization of straight vegetable oils at high injection pressures

We present results of high pressure spray characterization of Straight Vegetable Oils (SVOs) which are potential diesel fuel substitutes. SVO sprays are visualized at high injection pressures (up to 1600 bar) to study their atomization characteristics. Spray structure studies are reported for the first time for Jatropha and Pongamia vegetable oils, under atmospheric conditions. Jatropha and Pongamia SVO sprays are found to be poorly atomized and intact liquid cores are observed even at an injection pressure of 1600 bar. Non-Newtonian behavior of Jatropha and Pongamia oil is shown to be the reason for observed spray structure. (C) 2012 Elsevier Ltd. All rights reserved.

Spray characterization of gasoline-ethanol blends from a multi-hole port fuel injector

This work reports the measured spray structure and droplet size distributions of ethanol-gasoline blends for a low-pressure, multi-hole, port fuel injector (PFI). This study presents previously unavailable data for this class of injectors which are widely used in automotive applications. Specifically, gasoline, ethanol, and gasoline-ethanol blends containing 10%, 20% and 50% ethanol were studied using laser backlight imaging, and particle/droplet image analysis (PDIA) techniques. The fuel mass injected, spray structure and tip penetrations, droplet size distributions, and Sauter mean diameter were determined for the blends, at two different injection pressures. Results indicate that the gasoline and ethanol sprays have similar characteristics in terms of spray progression and droplet sizes in spite of the large difference in viscosity. It appears that the complex mode of atomization utilized in these injectors involving interaction of multiple fuel jets is fairly insensitive to the fuel viscosity over a range of values. This result has interesting ramifications for existing gasoline fuel systems which need to handle blends and even pure ethanol, which is one of the renewable fuels of the future. (C) 2012 Elsevier Ltd. All rights reserved.

Breakup and coalescence characteristics of a hollow cone swirling spray

This paper deals with an experimental study of the breakup characteristics of water emanating from hollow cone hydraulic injector nozzles induced by pressure-swirling. The experiments were conducted using two nozzles with different orifice diameters 0.3 mm and 0.5 mm and injection pressures (0.3-4 MPa) which correspond to Rep = 7000-26 000. Two types of laser diagnostic techniques were utilized: shadowgraph and phase Doppler particle anemometry for a complete study of the atomization process. Measurements that were made in the spray in both axial and radial directions indicate that both velocity and average droplet diameter profiles are highly dependent on the nozzle characteristics, Weber number and Reynolds number. The spatial variation of diameter and velocity arises principally due to primary breakup of liquid films and subsequent secondary breakup of large droplets due to aerodynamic shear. Downstream of the nozzle, coalescence of droplets due to collision was also found to be significant. Different types of liquid film breakup were considered and found to match well with the theory. Secondary breakup due to shear was also studied theoretically and compared to the experimental data. Coalescence probability at different axial and radial locations was computed to explain the experimental results. The spray is subdivided into three zones: near the nozzle, a zone consisting of film and ligament regime, where primary breakup and some secondary breakup take place; a second zone where the secondary breakup process continues, but weakens, and the centrifugal dispersion becomes dominant; and a third zone away from the spray where coalescence is dominant. Each regime has been analyzed in detail, characterized by timescale and Weber number and validated using experimental data. (C) 2012 American Institute of Physics. http://dx.doi.org/10.1063/1.4773065]

Dynamics and fracture of ligaments from a droplet on a vibrating surface

A droplet residing on a vibrating surface and in the pressure antinode of an asymmetric standing wave can spread radially outward and atomize. In this work, proper orthogonal decomposition through high speed imaging is shown to predict the likelihood of atomization for various viscous fluids based on prior information in the droplet spreading phase. Capillary instabilities are seen to affect ligament rupture. Viscous dissipation plays an important role in determining the wavelength of the most unstable mode during the inception phase of the ligaments. However, the highest ligament capillary number achieved was less than 1, and the influence of viscosity in the ligament growth and breakup phases is quite minimal. It is inferred from the data that the growth of a typical ligament is governed by a balance between the inertial force obtained from the inception phase and capillary forces. By including the effect of acoustic pressure field around the droplet, the dynamics of the ligament growth phase is revealed and the ligament growth profiles for different fluids are shown to collapse on a straight line using a new characteristic time scale.