Analysis of microparticle penetration into human and porcine skin: non-invasive imaging with multiphoton excitation microscopy

At the University of Oxford and PowderJect Pharmaceuticals plc, a unique form of needle-free injection technology has been developed. Powdered vaccines and drugs in micro-particle form are accelerated in a high-speed gas flow to sufficient velocity to enter the skin, subsequently achieving a pharmaceutical effect. To optimize the delivery of vaccines and drugs with this method a detailed understanding of the interactive processes that occur between the microparticles and the skin is necessary. Investigations to date of micro-particle delivery into excised human and animal tissue have involved image analyses of histology sections. In the present study, a series of investigations were conducted on excised human and porcine skin using the technique of Multi-Photon fluorescence excitation Microscopy (MPM) to image particles and skin structures post-penetration. Micro-particles of various size and composition were imaged with infrared laser excitation. Three-dimensional images of stratum corneum and epidermal cell deformation due to micro-particle penetration were obtained. Measurements of micro-particle penetration depth taken from z-scan image stacks were used to successfully quantify micro-particle distribution within the skin, without invasively disrupting the skin target. This study has shown that MPM has great potential for the non-invasive imaging of particle skin interactive processes that occur with the transdermal delivery of powdered micro-particle vaccines and drugs

Dispersion in oscillatory flows

The enhanced axial mixing which is caused by dispersion in oscillatory flows in some mass transfer devices may limit the reactor performance. This effect has provided the motivation for the present study in which oscillatory flow dispersion in a flat channel of large aspect ratio is investigated. The rate of spreading of a uniform slug of some passive tracer has been predicted using numerical and analytical techniques and the results have been verified experimentally. The numerical approach has used a finite difference time-marching method to obtain predictions for the channel concentrations. From the results, the dispersion coefficient (D) has been evaluated for Strouhal numbers of O.O1→0.2 and for mean Reynolds numbers of O.4→2OO at Schmidt numbers (Sc) O(1O³) . It has been concluded that under these conditions D varies as stroke squared. Unless the flow is not quasi-steady (i.e. if pulsatile Reynolds number α²O(l)) D is only a weak function of frequency. These predictions for the dispersion coefficient have been in excellent agreement with those of Watson (256). It has also been concluded from the numerical study that the phase of the velocity sinusoid at the instant of injection has a critical effect upon the form of the concentration evolution. An approximate analytical technique has been developed in which weighted mean cross-channel concentrations are defined. The wall concentration is expressed approximately using a Fourier series. This procedure leads to ordinary differential equations for the axial moments. When the axial variance of mean concentration and the dispersion coefficient were computed in this way for quasi-steady flows good agreement was obtained with the numerical work. Simple opto-electronic gauges have been developed to measure mean cross-channel concentrations. The sensors have been used to obtain experimental data for the dispersion coefficient of a furrowed channel mass transfer device using slug stimulus techniques. Experimental investigations of dispersion in oscillatory flows in a flat channel using these gauges has produced values for D which are in agreement with the theoretical predictions for quasi-steady flows.</p

A ballistic study of micro-particle penetration to the oral mucosa

This paper describes the results of an investigation into the impact of model micro-particles to ex vivo buccal mucosa (the cheek) of pigs and beagle dogs. The work is aimed at optimizing a unique form of pharmaceutical delivery. The pharmaceutical is formulated into particle form and accelerated toward the target of skin or mucosa by using a gas jet. In this study, research devices designed using analytical, computational (computational fluid dynamics) and experimental methodologies were used to deliver particles at uniform and predetermined velocities. These velocities were confirmed using light obscuration, pressure measurement and particle image velocimetry methods. Polystyrene, glass, stainless steel and gold micro-particles at a range of size distributions were used for the payloads for injection using these devices. Injection occurred with a wide range of impact conditions into fresh canine (dog) and porcine (pig) buccal mucosa. Final positions of the particles were determined from histological sections and the results analyzed in relation to the known particle impact parameters of size, density and velocity. The experimental results are collated using an empirical term, based on particle impact parameters. Comparison is then made to a semi-empirical penetration model. This is done by analysing the penetration results in terms of each particle impact parameter. Analysis shows that this model fits the experimental data well when reasonable estimates of the tissue mechanical properties are chosen. (C) 2002 Elsevier Science Ltd. All rights reserved

Characterization of powdered epidermal vaccine delivery with multiphoton microscopy

Multiphoton laser scanning microscopy (MPLSM) has been adapted to non-invasively characterize hand-held powdered epidermal vaccine delivery technology. A near infrared femtosecond pulsed laser, wavelength at approximately 920 nm, was used to evoke autofluorescence of endogenous fluorophores within ex vivo porcine and human skin. Consequently, sub cellular resolution three-dimensional images of stratum corneum and viable epidermal cells were acquired and utilized to observe the morphological deformation of these cells as a result of micro-particle penetration. Furthermore, the distributional pattern of micro-particles within the specific skin target volume was quantified by measuring the penetration depth as revealed by serial optical sections in the axial plane obtained with MPLSM. Additionally, endogenous fluorescence contrast images acquired at the supra-basal layer reveal cellular structures that may pertain to dendritic Langerhans cells of the epidermis. These results show that MPLSM has advantages over conventional histological approaches, since three-dimensional functional images with sub-cellular spatial resolution to depths beyond the epidermis can be acquired non-invasively. Accordingly, we propose that MPLSM is ideal for investigations of powdered epidermal vaccine delivery

Multiphoton high-resolution 3D imaging of Langerhans cells and keratinocytes in the mouse skin model adopted for epidermal powdered immunization

Langerhans cells (LCs) can be targeted with DNA-coated gold micro-projectiles ("Gene Gun") to induce potent cellular and humoral immune responses. It is likely that the relative volumetric distribution of LCs and keratinocytes within the epidermis impacts on the efficacy of Gene Gun immunization protocols. This study quantified the three-dimensional (3D) distribution of LCs and keratinocytes in the mouse skin model with a near-infrared multiphoton laser-scanning microscope (NIR-MPLSM). Stratum corneum (SC) and viable epidermal thickness measured with MPLSM was found in close agreement with conventional histology. LCs were located in the vertical plane at a mean depth of 14.9 mum, less than 3 mum above the dermo-epidermal boundary and with a normal histogram distribution. This likely corresponds to the fact that LCs reside in the suprabasal layer (stratum germinativum). The nuclear volume of keratinocytes was found to be approximately 1.4 times larger than that of resident LCs (88.6 mum3). Importantly, the ratio of LCs to keratinocytes in mouse ear skin (1:15) is more than three times higher than that reported for human breast skin (1:53). Accordingly, cross-presentation may be more significant in clinical Gene Gun applications than in pre-clinical mouse studies. These interspecies differences should be considered in pre-clinical trials using mouse models