26.1. Introduction

Particle-mediated delivery involves coating materials onto the surface of dense sub-cellular sized (0.5-5 mm) particles and accelerating the particles to sufficient velocity to penetrate target cells. The technique was invented by Sanford and Wolf at Cornell University (1) to transfer DNA into intact plant cells (2), and was further developed into an effective process for producing genetically engineered crop plants by several groups (reviewed in 3). Subsequent work has shown that this method is generally applicable for transferring materials including DNA, RNA, proteins, peptides and pharmacological compounds into a wide variety of tissue and cell types in vivo, ex vivo, or in vitro (reviewed in 4).

The topic of particle-mediated gene transfer to epidermis as a means of nucleic acid immunization is specifically addressed in an accompanying chapter. This chapter describes procedures for preparation of the necessary materials (see Note 1). The procedures are divided into two sections: bead preparation and tube preparation. The first section describes procedures for making a slurry of DNA-coated gold particles, and the second section procedures for loading the DNA-coated particles into "cartridges."

Particle-mediated gene transfer has primarily employed gold particles as carriers. Particles in the sub-cellular size range must be dense to achieve the momentum necessary for adequate penetration. Among the materials with sufficient density (e.g., gold, iridium, platinum, tungsten) gold is preferred because of its low chemical reactivity and low toxicity. Moreover, high purity gold powders in the desired size range are commercially available.

Powder injection is a conceptually distinct technology that is, in several aspects, complementary to particle-mediated delivery. Rather than using dense sub-cellular sized carrier particles, as in particle-mediated delivery, powder injection employs larger low-density particles that can be formulated without a dense carrier. Sarphie et al. (5) demonstrated the systemic distribution of inulin administered dermally in the form of low-density (1.5 g/cm 3) particles. Particles in the 30-50 μm size range were required, thus delivery was likely extra-cellular; however, significant systemic bio-availability was achieved. These results suggest that powder injection is suitable for delivery of conventional pharmaceuticals, conventional vaccines, and other substances where extra-cellular delivery is effective and cellular uptake of the delivered material is efficient. Particle-mediated delivery, on the other hand, is an effective means of direct intra-cellular delivery. The use of powder injection for nucleic acid immunization is under investigation.

A number of parameters that influence particle-mediated gene delivery can be readily adjusted to optimize performance in a particular system. These parameters include the accelerating force, the size of the particles, the number of particles per target site, the distribution of particles within the target, and the amount of DNA loaded onto the particles. Manipulation of these parameters provides versatility, allowing adaptation of the method to target tissues with widely differing physical characteristics. A number of conveniently assayed reporter genes ( e.g., human growth hormone, ~-galactosidase, green fluorescent protein) are available that can be used to optimize parameters for a specific system. Reporter genes that can be assayed histologically are especially valuable for identification of transfected cells within a tissue as well as measurements of transfection efficiency.

Several devices have been developed to accelerate the coated particles; these include

1. the original Biolistic Device of Sanford and Wolf (2),

2. the PDS-1000/He device and Helios Gene Gun manufactured by Bio-Rad Laboratories Inc. (Hercules, CA),

3. a hand-held helium-driven device designed by John Sanford and Stephen Johnston (6), and

4. the Accell electric-discharge (7) and helium-powered (8) devices designed by Dennis McCabe and co-workers at Agracetus, Inc.

5. A disposable device (PowderJect), described by Sarphie et al. (5) for powder injection, offers several attractive features in terms of practical clinical application of the technology.

6. A device that combines these features of the PowderJect device with the delivery capabilities of the Accell helium device is currently under development.

This chapter describes procedures for using the helium-driven Accell device (8). A similar device, the Helios Gene Gun, and associated equipment and supplies are commercially available from Bio-Rad. The Helios device was developed in collaboration with Powderject Vaccines Inc. (Madison, WI) to provide devices for the life sciences research community. Particle-mediated gene transfer is a proprietary technology covered by patents held by PowderJect Vaccines, Inc. (previous names or affiliates include Geniva Inc., Auragen Inc., and Agracetus Inc.) and E. I. DuPont de Nemours & Co. (Wilmington, DE). Devices from Bio-Rad that implement this technology are licensed for research purposes only, and are not intended or approved for clinical use.