Biological delivery systems have long been the focus of much research due to the intense interest in introducing a variety of biological substances into a target. Such substances include nucleic acids, genetic constructs, drugs, hormones, proteins, biological stains, etc. Targets include cells, germ cells, plants, microbes, animals, organelles, tissues, etc. Diverse applications for biological delivery systems are known as is illustrated in The Pharmacological Basis of Therapeutics, Goodman and Gilman, Chapter 1, pages 1-28 (1970).
A number of delivery systems for biological substances are available. Among those options are oral ingestion, nasal sprays, pulmonary absorption, parenteral injection, transdermal or topical delivery. However, there is a desire to extend the accessibility and release (bioavailability) of a biological substance to its target. For example, genetic material can be delivered into a target. Existing technologies for introducing genetic material into living cells involve a number of systems some of which utilize biological vectors such as retroviruses or Agrobacterium to accomplish gene transfer. Others rely on chemical, e.g., calcium phosphate precipitation, polyethylene glycol, lipofection, or liposomes or physical means such as injection, microinjection or electropotation or particle bombardment to facilitate DNA transfer across cell membranes. These technologies are reviewed in Klein et al., Bio/Technology, 10: 286-291 (March 1992).
Particle bombardment also referred to as Biolistic.RTM. is a physical method for gene delivery which involves accelerating DNA-coated particles (microprojectiles) directly into cells. The Biolistic.RTM. process is described in U.S. Pat. No. 4,945,050 which issued to Sanford et al. on Jul. 31, 1990; Sanford, Biomedical Engineering: Opening New Doors, Proceedings of the 1990 Biomedical Engineering Society, Milurlecky and Clarke (Eds.) (1990); Klein et al., Bio/Technology, 10: 286-291 (March 1992). These microprojectiles must have sufficient mass to acquire the necessary kinetic energy to penetrate the particular cell sought to be penetrated and they must have integrity sufficient to withstand the physical forces inherent in the the process. The microprojectiles are described as being inert particles including ferrite crystals, gold, tungsten, spheres as well as particles of low density such as glass, polystyrene and latex beads. Biological particles can also be used. The biological substance to be transported is coated, impregnated or otherwise operably associated with solid particle to be introduced into the cell.
Particle-mediated transformation of somatic cells is described in PCT International Application having International Publication Number WO91/0035 published on Jan. 10, 1991. In this process an electric discharge particle acceleration apparatus such as the Accell.RTM. particle acceleration apparatus is used to inject very small particles of gold or other dense material carrying genetic constructs coated on them into the living cells of animals.
The ability to introduce foreign genes into the germ line and to obtain successful expression is an important aspect of genetic engineering. Such transgenic animals can be used for production of pharmaceutical proteins, disease models, improved animal health, appearance and stamina, etc.
Methods for introducing foreign genes into the germ line are limited at this time. Such methods include microinjection and retroviral transfection. Microinjection of cloned DNA directly into the pronucleus of a fertilized mouse egg has been the most widely used method for generating transgenic mice. However, the injected DNA-does not always integrate into the host genome.
One alternative considered by researchers to overcome this problem is to exploit the ability of spermatozoa to introduce foreign DNA into eggs at fertilization. Lavitrano et al., Cell 57: 717-723 (1989) purported that they were able to use sperm to transfer plasmid DNA into eggs. However, several attempts to duplicate this work have been unsuccessful to date, Brinster et al., Cell 59: 239 (1989).
Another alternative which has not been considered is the use of particle bombardment to transfer DNA directly into sperm. The method is limited because useful particles of the appropriate size for delivery into sperm have not been available. Indeed, if such particles were available then the efficiency of this method for transforming other targets of biological importance such as plants, organelles, cells, animals, organoids and tissues would be improved. In addition, the method could be extended to transform germ cells, microbes, microalgae, etc.
Recently, particles having a substantially pure carbonaceous surface were discovered in 1991 as a by-product of fullerene production, Iijima, Nature, Vol. 259, pages 56-58 (1991). Most recently, Ruoff et al., Science, Vol. 259, pages 346-348 (Jan. 15, 1993) have described novel particles in which single crystal metals were encapsulated in particles having a substantially pure carbonaceous surface. Specifically, single domain .alpha.-LaC.sub.2 small crystals were encapsulated inside particles having a substantially pure carbonaceous surface. To date no applications have been reported for these particles having a substantially pure carbonaceous surface and the like.
Surprisingly and unexpectedly, it has been found that such particles can be used not only to transform targets by means such as particle bombardment, but also such particles can be used to deliver a biological substance into a target as part of a biological delivery system. Such findings have not heretofore been reported.