Characteristics of a micro-biolistic system for murine immunological studies.

With an advanced computational fluid dynamics (CFD) technique, we have numerically developed and examined a micro-biolistic system for delivering particles to murine target sites. The micro-particles are accelerated by a high speed flow initiated by a traveling shock wave, so that they can attain a sufficient momentum to penetrate in to the cells of interest within murine skin (or mucosa). In immunization application, powdered vaccines are directly delivered into the antigen presenting cells (APCs) within the epidermis/dermis of the murine skin with a narrow and highly controllable velocity range (e.g., 699+/-5.6 m/s for 1.8 microm modeled gold particles) and a uniform spatial distribution over a diameter of approximately 4 mm target area. Key features of gas dynamics and gas-particle interaction are presented. Importantly, the particle impact velocity conditions are quantified as a function of: stand-off distance (2-15 mm), driver gas species (air/helium mixtures), particle density (1,050 kg/m3 and 19,320 kg/m3) and particle size (1-5 microm for gold particles and 10-50 microm for less dense particles, respectively). The influential parameters--representative of immunotherapeutic (e.g., DNA vaccination) and protein (e.g., lidocaine) biolistic applications--are studied in detail.

Identification of second harmonic optical effects from vaccine coated gold microparticles.

This study investigates the optical effects observed from uncoated and protein vaccine coated gold microparticles while imaging with two-photon excitation in the Mie scattering regime. When observed with time correlated single photon counting fluorescence lifetime microscopy, the emission from the gold microparticles appeared as an intense instrument-limited temporal response. The intensity of the emission showed a second-order dependence on the laser power and frequency doubling of the emitted light was observed for fundamental light between 890 and 970 nm. The optical effect was attributed to two-photon induced second harmonic generation. The vaccine coated gold microparticles had a much weaker second harmonic signal than the uncoated gold microparticles. Chemical analysis of the surface of the gold microparticles revealed that the vaccine coating decreases the surface charge thereby diminishing the observed second harmonic signal. These optical properties can be exploited to identify both the location of the protein vaccine coating as well as the gold microparticles in vitro and potentially to investigate the vaccine delivery kinetics in vivo.

Development of a dye-ligand affinity membrane for the separation of bovine serum albumin.

This study reports the development and testing of a dye-ligand membrane. A synthetic dye called Cibacron Blue F3G-A was utilized as the capturing ligand. Bovine serum albumin acted as the target protein. To immobilize the dye molecules, a microfiltration membrane (Versapor) was used as the supporting matrix. The pore size rating of this membrane was reported to be 0.45 micron. Two different methods of dye immobilization were tested. In the first method, the ligand was directly attached to the membrane, whilst in the second method, a spacer molecule was first coupled to the matrix and then dye coupling was carried out. The chemical stability of the adsorbed dye was studied by subjecting the dye-membranes to appropriate desorption protocols. As a model affinity system, the adsorption of bovine serum albumin onto the dye-membranes was thoroughly investigated. The equilibrium behavior and the kinetic parameters of the BSA-dye interaction were determined experimentally.

Transdermal and transmucosal powdered drug delivery.

High-velocity powder injection is a promising new drug-delivery technique that provides needle- and pain-free delivery of traditional drugs, drugs from biotechnology such as proteins, peptides, and oligonucleotides as well as traditional and genetic vaccines. The energy of a transient helium gas jet accelerates fine drug particles of 20 microns-100 microns diameter to high velocities and delivers them into skin or mucosal sites. This review describes the configuration and operating principles of devices that accelerate the particles, the required properties of the particles, the characteristics of the target tissues, and features of the developmental test methods. Preclinical and clinical results that best characterize the technology and introduce its potential as a drug-delivery platform are presented.

Modelling of the flow-through dye-ligand affinity membrane process using bovine serum albumin as the model protein.

This paper presents the application of a mathematical model for the prediction of the performance of a dye-ligand affinity membrane. Cibacron Blue F3G-A was utilised as the capturing ligand and bovine serum albumin as the target molecule. The dye molecules were immobilised covalently via spacer molecules (polyethlenimine) onto the pores of a microfiltration membrane with a pore size rating of 0.45 micron. Performance analysis of the affinity membranes was achieved by studying the resulting breakthrough curves, when BSA solutions were passed through the system. In the mathematical model, phenomena such as convection, axial dispersion, and adsorption rate of adsorbate were incorporated so that a clearer interpretation of the experimental data could be obtained. Numerical analysis was used to solve the governing partial differential equations.

Oral PowderJect: a novel system for administering local anaesthetic to the oral mucosa.

OBJECTIVE: To assess the feasibility of using an Oral PowderJect (OPJ) to safely deliver a dose of dry powdered anaesthetic to the oral mucosa, producing an analgesic effect. DESIGN: Single centre: Part 1. An open, non-randomised safety study to check for mucosal damage; Part 2. A double blind sham controlled study to test the anaesthetic effect. SETTING: General practice. SUBJECTS: Adult, healthy volunteers (4 male, 10 female). MATERIALS AND METHODS: Part 1. An OPJ was used to deliver powdered lidocaine hydrochloride to the mucosal surface which was then checked visually for damage. Part 2. An OPJ containing lidocaine hydrochloride (active) or an empty OPJ (sham) was fired at the oral mucosa. The treated area and an untreated (control) site were probed with the back end of a dental needle. RESULTS: The OPJ delivery caused no visible mucosal damage. The median VAS score for pain on blunt probing was 10 for the OPJ active sites. This was significantly lower than the median VAS score for the sham sites at 30 (P = 0.0033) and the control sites at 58 (P < 0.0001). CONCLUSIONS: The OPJ can safely deliver powdered lidocaine hydrochloride to the oral mucosa without causing tissue damage. The OPJ delivery of powdered lidocaine hydrochloride can significantly reduce the pain from a blunt needle probe at 1 minute post delivery.

Effect of oscillatory flow on the performance of a novel cross-flow affinity membrane device.

This paper presents the results of an investigation into the effect of oscillatory flow in a membrane-based affinity contactor. This device was designed to accommodate a tubular affinity membrane, and the flow direction of working fluid was tangential to the surface of the membrane. Cibacron Blue F3G-A was utilized as the capturing ligand and bovine serum albumin as the target molecule. The dye molecules were immobilized covalently via spacer molecules (polyethylenimine) onto the pores of a microfiltration membrane with a pore size rating of 0.45 micron. Bovine serum albumin was pumped through the annular space between the concentric screw-threaded insert and the tubular membrane in oscillatory flow with a mean flow component. The effects of pulsation frequency and stroke length were investigated. It was found that, as a result of the pulsatile flow, the protein recovery was increased by a factor of 2. To make the interpretation of the results easier, various dimensionless groups were defined specifically for this system and the experimental data were reported in terms of these groups.

Mammalian cell damage in a novel membrane bioreactor.

The experimental study has assessed a novel membrane bioreactor for mammalian cell culture. In the absence of a gas phase, the key features of cell damage associated with laminar and turbulent flow have been identified. The bioreactor employs a dimpled membrane in order to enhance transverse mixing in a narrow channel, but a fall in viable cell density has been observed at Reynolds numbers above Re = 83. In the laminar flow regime wall shear is the critical mechanism and an accurate calculation of shear rate in a complex channel has been achieved using the Reynolds analogy. Flow generating a wall shear rate in excess of 3000 s(-1) has been shown to cause damage. Power dissipation measurements have been used to distinguish between laminar and turbulent flow and also to predict Kolmogorov eddy lengths. An additional turbulent bulk stress damage mechanism at higher Reynolds numbers (Re > 250) results in a very rapid fall in viable cell density.

Enhanced filtration using flat membranes and standing vortex waves.

The performance of membrane filters is limited by concentration polarisation and membrane fouling. High shear rates reduce these effects but can damage delicate cells and proteins in biological fluids. Secondary flows to promote fluid mixing, and hence enhance filtration, can be highly effective, but usually carry the penalty of complexity. Oscillatory flow over furrowed or dimpled membrane surfaces (vortex mixing) has been shown to be very efficient in the microfiltration and oxygenation of blood, but carries the added complexity of oscillatory flow and the need to thermoform the membranes. To allow the use of asymmetric or rigid membranes, a new form of vortex mixing has been studied, in which the membranes are flat and form parallel channels. Feed flow is oscillatory and passes back-and-forth across ladder-like flow deflectors to generate standing vortex waves. Flow visualisation studies and numerical calculations of standing vortex waves (SVW) were undertaken to help optimise flow parameters and flow deflector geometry. Flat plate membrane ultrafilters were built and evaluated for different geometries of flow deflector, using solutions of bovine serum albumin (BSA) as the test fluid. Very high filtration rates were measured with BSA concentrations ranging from 1 to 24%. A SVW ultrafilter was compared with a Millipore Pellicon ultrafilter using BSA as the test fluid. Filtration flux was similar at low concentrations of BSA, but was greater by a factor of 4.5 in the SVW device at high concentrations of BSA.

A novel membrane bioreactor for microbial growth.

A novel membrane bioreactor, previously assessed for its gas transfer characteristics, was used in various size and membrane configurations for the growth of the strictly aerobic bacterium Pseudomonas aeruginosa. The bioreactor was found to readily support growth, and the initial growth rates showed the previously demonstrated enhanced effect in gas O2 mass transfer of the dimpled membrane bioreactor over flat membrane bioreactors. The production of a secondary metabolite by a Pseudomonas sp. following growth was demonstrated, as was the biotransformation of a nitrile by Nocardia rhodochrous with the removal of the biotransformation products across a membrane. The potential of the bioreactor, in terms of other applications in the field of biotechnology, is discussed.

Quantitative optical determination of the viability of platelet concentrates.

An optical method to assess platelet viability in platelet transfusion concentrates has been developed which is based on the observation that healthy, discoid platelets transmit more light in shear flow than aged, spherical cells. Using a specially designed glass channel, the rise in transmission of light through platelet samples with agitation is referenced against a measurement of transmission without flow. Referencing the measurements may minimize the effects of red blood cell contamination, as well as other variations among platelet packs such as plasma characteristics and plastic container variability. At an optimal frequency of agitation, the ratio of the transmission of light with agitation to that of the sample at rest is found to correlate highly with the concentration of discoid platelets, r = 0.92 to 0.95 (P less than 0.001), for light sources including a HeNe laser and two LEDs. Using this index, the discoid platelet counts of twelve platelet packs are estimated and compared with manual counts. The mean difference between the two methods was 0.009 x 10(9) discoid platelets ml-1 with a standard deviation of 0.11 x 10(9) cells ml-1. We conclude that the method may be applied to the development of a non-invasive device to monitor the concentration of viable, discoid platelets in storage.

Gas transfer characteristics of a novel membrane bioreactor.

We show the design features of a membrane bioreactor based on pulsatile flow across dimpled membranes. Results show an enhanced mass transfer of air of at least five-fold magnitude as compared with flat membranes. An increased working volume form 20 mL to 120 mL reduced the k(L)A at a given Reynolds number because of axial mixing of fluid from the deoxygenated end chamber. The bioreactor was used to supply air to a hybridoma mammalian cell line, and the calculated oxygen uptake showed that high-density cultures could be maintained in a 20 mL, single-dimpled cultures could be maintained in a 20 mL, single-dimpled membrane system. Indirect aeration of a 2 L continuous stirred tank reactor, by a double-membrane system, showed that air could be supplied to mammalian cells at cell densities of approximately 4 x 10(6) /mL.

Comparative study of reaction kinetics in membrane and agarose bead affinity systems.

Compared to conventional agarose bead affinity supports, a microporous nylon membrane exhibits greatly improved reaction kinetics as quantified in the reaction between gamma-globulin and immobilised protein A. The improvement is only observed when the solution of gamma-globulin is forced through the membrane pores. In the absence of flow in the pores, it is possible to relate approximately the rate of uptake onto either type of affinity support to independently determined diffusion coefficients. In the presence of flow, the reaction rate is similar for membranes having 0.45 and 3.0 microns diameter pores, and considerably smaller than predicted by the Smoluchowski formula.

A high efficiency membrane separator for donor plasmapheresis.

The shortfall in human plasma for therapeutic use can be met satisfactorily only by donor plasmapheresis. Membrane blood plasma separators of conventional design work poorly at low rates of blood flow, and performance decreases rapidly as the membrane becomes fouled. To overcome these problems, membrane separators have been developed that employ vortex mixing to prevent blood cells obstructing the membrane pores. These separators consist of a single blood channel 160 mm long by 62.5 mm wide. The two membranes that act as boundaries for the blood channel are thermoformed to provide a closely packed array of outward facing dimples. The membranes are seated on rigid support plates, and flexible diaphragms are attached to each end of the blood channel. The diaphragms were oscillated in antiphase at 3 to 6 Hz, with a stroke volume of 2.8 ml. In 24 clinical trials, mean plasma filtration rate was 18 ml/min at a blood inlet flow of 48 ml/min. In 10 experiments using bovine blood, mean plasma filtration rate was 35 ml/min at a blood inlet flow of 90 ml/min. Little reduction in performance was observed over 80 min. The membrane area was 200 sq cm, which gave a filtration velocity of 1.75 L/min/m2, higher by a factor of five than is reported for hollow fiber or flat plate designs.

Separation of plasma from whole blood by membrane filtration in oscillatory flows.

This paper describes the performance of a microfiltration plasmapheresis unit operating with reversing oscillatory flows. The device consists of a flat channel duct between cellulose nitrate membranes and was used to extract plasma from anticoagulated fresh whole bovine blood. Measurements were made of plasma flux, haematocrit concentration, haemolysis and protein sieving coefficients. The effects on plasma flux are reported for alterations in the stroke and frequency of flow pulsations, transmembrane pressure, membrane properties and blood throughput. It was found that the imposition of oscillatory flows enhanced the plasma extraction rate by a factor of 3, producing about 0.9 litre/min/m2 membrane.

Extracorporeal oxygen and CO2 transfer of a polypropylene dimpled membrane lung with variable secondary flows: partial bypass in the dog.

In vivo gas transfer performance, during veno-venous bypass, is presented for one form of the Oxford membrane lung in which vortex mixing is induced in blood flow across a dimpled polypropylene membrane. Good agreement has been found with in vitro data presented in an earlier paper. Two experiments were extended to attain total extracorporeal CO2 removal with apnoeic diffusion oxygenation to motionless lungs. Measurements of arterial oxygen partial pressure during apnoea suggest that the time-constant for equilibration of alveolar nitrogen partial pressure with that on the gas side of the membrane lung is of the order of 100 min.

Oxygen and CO2 transfer of a polypropylene dimpled membrane lung with variable secondary flows.

Gas transfer performance is presented for one form of the Oxford membrane lung in which vortex mixing is induced in blood flow across a dimpled polypropylene membrane. Dimensional analysis has been used to define the parameters characterizing mass transfer in the device, and of three fluid mechanical parameters: Reynolds number based on peak pulsation velocity, Strouhal number, and ratio of mean to oscillatory flows, only the first has been found to affect mass transfer significantly in the ranges studied. For oxygenation, rated flows in excess of 5 l min-1 m-2 are measured. A new definition is presented for a rated flow for extracorporeal CO2 removal and values in excess of 1.2 l min-1 m-2 are obtained.

Initial in vitro evaluation of a pediatric vortex-mixing membrane lung.

A new design for a pediatric membrane lung is described in this paper. The lung consists of eight blood compartments, each having six U-shaped blood channels, with microporous PTFE membranes supported on rigid plates in such a way that the membranes form furrowed blood channels. Two rolling diaphragm pumps are attached to the open ends of the U-shaped blood channels; these pumps are operated in antiphase. Mean flow is provided by a roller pump placed at the inlet end of the membrane lung. Pulsatile blood flow within the blood channels produces successive vortex formation and ejection, leading to good blood mixing and high efficiency in gas transport. The design of the rolling diaphragm piston pumps ensures that the blood prime volume is low (280 ml), and the grouping of the pumps at one end of the oxygenator allows the driving mechanism to be simple and compact. The relatively wide blood channels (minimum width 0.5 mm) and vortex mixing make priming the membrane lung particularly easy. The membrane area is 0.39 m2. Preliminary performance testing of the pediatric membrane lung was undertaken by pumping blood around a circuit containing a roller pump, the membrane lung, and a bubble oxygenator (to adjust the blood gases at the inlet to the membrane lung). In five such experiments it was shown that the membrane lung transferred 80 ml O2/min and 120 ml CO2/min at a blood flow rate of 1.5 L/min.