Stock material or miscellaneous articles – Coated or structually defined flake – particle – cell – strand,... – Particulate matter
Reexamination Certificate
2001-05-02
2004-03-16
Kiliman, Leszek (Department: 1773)
Stock material or miscellaneous articles
Coated or structually defined flake, particle, cell, strand,...
Particulate matter
C428S403000, C428S404000, C428S407000, C428S329000, C428S690000, C428S900000, C427S127000, C427S216000, C427S221000, C435S173100, C436S526000
Reexamination Certificate
active
06706394
ABSTRACT:
BACKGROUND OF THE INVENTION
When conducting genetic manipulations or other treatments of cells or tissue, it is often necessary to partially or completely penetrate the cell walls and/or membranes with a biological or other agent. This penetration is necessary in order to achieve a desired effect on the cell wall and/or internal cellular elements such as the cytoplasm, nucleus, plastids, chromosomes, plasmids, etc. The objective of such a procedure may be, for example, the destruction of selected substances or the production of new or improved biological characteristics. These procedures can be used to modify a plant, animal, or microbe to improve, for example, growth rate, disease resistance, or protein production. Other applications include the tagging of cells for tracking and identification, or the micromanipulation of cells by in situ rotation or displacement in space.
In genetic research, for example, such methods are used to penetrate tissue and cells with particles precoated with DNA encoding genes of interest; cell penetration is followed by DNA delivery into the cell nucleus or organelle. To reach the intracellular space and then the cell subcellular structure, the particles must traverse formidable cell walls and membranes. Because these cell walls are difficult to penetrate, the particles carrying the DNA are driven into the cells by the force of an explosive or an electrical discharge so that the kinetically driven particles smash into the target tissue. Even then, in order to have the necessary kinetic energy for penetration of certain targets and to certain depths, the particles must be several micrometers in diameter. Thus, the implantation process results in appreciable cell damage due to the impact of the particles and/or due to sonic concussion from the particle-propelling discharge. Some cell tissue, drawing upon its natural strength, may recover from this trauma sufficiently to integrate the newly delivered genetic material into its chromosomes; however, a significant percentage of the tissue is unable to do so.
These prior methods of delivering particles to cells also lack sufficient control over particle size distribution, particle coating quality, and the velocity and direction of travel of the particles, resulting in a lack of predictability and reproducibility of the particle delivery technique. The prior delivery techniques are further disadvantaged because they require that the target tissue be maintained in a vacuum which removes moisture from the treated tissue contributing to tissue degradation. Moreover, the apparatus for performing the implantations requires time-consuming set up prior to each implantation cycle and is cumbersome to clean after same so that the throughputs of the apparatus are relatively low.
Other methods employed or suggested for direct gene delivery to cells include the use of microlasers, microbead vortexing, electrofusion, chemical fusion, microinjection, and electroporation. Such techniques all rely on increasing the permeability of the tissue cells by physically, chemically, or electrically disrupting cell walls and/or membranes temporarily; exogenously added DNA may then enter the cell through the temporary ruptures. Some of these methods, including microinjection and fusion of preselected protoplasts or subprotoplasts, require working at the single cell level. This necessitates micromanipulation of the cells, often involving immobilization by agarose plating or pipette suction. Such micromanipulations must be carried out with a microscope placed in the sterile environment of a laminar flow hood, which can be very cumbersome. Also, controlled fusion, for example in the production of somatic hybrids, requires bringing the fusion partners into close proximity which is technically difficult to accomplish. Another bottleneck in plant and other genetic transformation systems is the relative inefficiency of selection following gene transfer. A means to enrich for penetrated cells or organelles prior to, or in place of, selection by use of antibiotics, herbicides, osmotics or toxins would greatly improve the efficiency of a transgenesis system.
A number of magneto-mechanical systems have recently been devised whose purpose is to deliver certain reactive substances to a target site using sharp-tipped microparticles as carriers which penetrate the target sites such as pollen, cells, organisms, etc. to deliver these substances in singular or multiple sequential entries by desorption from the microparticles to cause a change in the target site, such as altering a genetic trait or curing a disease.
U.S. Pat. No. 5,516,670 discloses a method for delivering particles into cellular specimens by means of a non-uniform, convergent magnetic field and is incorporated herein by reference.
There still exists a need for particles which are designed to effectively perform each of a variety of tasks. For example, particles can serve as “payload carriers” such as to either deliver or extract one or more substances of interest to or from target sites. In another example, particles can “mark” the target for detection and/or counting purposes. Particles themselves may be the therapeutic agent, as in heat therapy. In addition, the subject particles can be furnished with properties which enable, or enhance, chemical interactions to achieve the desired treatment goals. Because of the variety of treatments required such as genetic alteration of a cell via DNA coupling or gene splicing or addressing different target sites such as pollen, cells, meristems, or human cancers, tumors, lymph nodes or nerve endings, a wide variety of microparticles are needed in terms of length, width, tips, geometries, and basic materials.
BRIEF SUMMARY OF THE INVENTION
The subject invention provides methods and apparatuses for the manufacture of magnetizable carrier particles (“micromagnets”). In addition, the subject invention pertains to particles having one or more of a variety of particle configurations and/or functional features. These particle configurations and/or functional features can be tailored to achieve one or more desired missions. The subject invention also pertains to methods and apparatuses for the delivery of particles to target materials, in order to accomplish one or more of a variety of missions.
In a specific embodiment of the subject invention, acicular and other particles with a lengthwise dimension that are uniform and homogenous in their geometry are manufactured and provided with magnetizations. In this way, predictable mechanical force responsivity can be achieved when these particles are subjected to an external magnetic field gradient. Preferably, saturation magnetization can be provided to the particles in order to yield optimal force for a particular particle size when inserted into a magnetic field gradient.
In another embodiment of the subject invention, the size, length, cross-sectional area, and/or shape and geometric contours of the particles, including asymmetry of mass, are selected such that the particle is optimally related to the target body. Accordingly, the particle's configuration can vary based on the target's size, shape, resistance against penetration, and other characteristics of the particle. For example, the subject particles can be provided with sharp “tips” at one or both ends. These sharp tips enable the particles to exert powerful pressures (force per unit area) at the point of contact between particle tip and target body, enhancing the ability of the particles to breach a protective wall of the target and to enter its interior space. One such embodiment involves providing a particle with a sharp “tip” at one end and a broader, nail-head-like tail at the other end, such as to enable partial penetration of the particle and, if desired, subsequent retraction of the particle. Such a particle can be retracted using, for example, a tape-like backing which can adhere to the particle's broader tail.
In another specific embodiment, the surface of the particles can be shaped such as to attract and/or accommodat
Kuehnle Adelheid
Kuehnle Manfred R.
Kiliman Leszek
Saliwanchik Lloyd & Saliwanchik
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