Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2001-01-10
2004-08-03
Beisner, William H. (Department: 1744)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S459000, C435S470000, C435S285100, C435S285200, C435S285300, C604S272000
Reexamination Certificate
active
06770480
ABSTRACT:
This invention relates to ways of transferring materials into cells, and also to a microneedle array.
There are many times when it is necessary to transfer materials into cells, for example nucleic acids or nucleic acid constructs, such as vectors or plasmids, etc. have to be transferred into a cell for the purposes of genetic manipulation. Furthermore, chemicals may also need to be transferred into cells, e.g. nucleotides or stains, and chemicals to affect the physiology of a cell. A number of chemical and mechanical processes have been developed to convey materials into cells. These techniques include:
1. direct microinjection—a needle is inserted into a cell and material expelled through the needle;
2. electroporation—the cell membrane is made permeable to some molecules by application of a high voltage shock;
3. biolistics—tungsten or gold particles are coated with the substance desired to be introduced and are shot into the cell;
4. calcium phosphate co-precipitation—cells absorb calcium phosphate, and if DNA/other material co-precipitates with the calcium phosphate it is also taken into the cell;
5. mediated transformation (via liposome. viral, or bacterial vectors): and
6. protoplast transformation.
An aim of one aspect of the present invention is to use a new material to assist in the transfer of substances to cells.
An aim of another aspect of the invention is to provide an improved way of providing small volumes of a substance.
Direct microinjection involves the insertion by a microneedle of DNA directly into the nucleus of individual cells. A glass micropipette linked to a micromanipulator is used to inject 10
−8
-10
−7
&mgr;l of material, typically a solution of DNA fragments, into cell nuclei. “Hits” are almost certain, given considerable operator expertise, but the technique is laborious and cannot be applied to a large number of cells.
According to a first aspect the invention comprises a method of transferring a substance into the cell.
Preferably resorbable or bioerodable porous silicon is used.
In one embodiment a microneedle that comprises at least a region of porous silicon is used, or an array of such microneedles is used.
According to a second aspect the invention comprises a microneedle (or microneedle array) comprising porous silicon.
According to a third aspect the invention comprises a vehicle for transferring material into a cell, the vehicle comprising, at least in part, porous silicon, and material to be transferred into the cell.
Preferably the porous silicon is resorbable. The vehicle may comprise a porous silicon biolistic bullet. The vehicle may comprise a substance which in use will co-precipitate with a co-precipitate substance that is taken into the cell. The vehicle may comprise an electrically-conducting bioactive porous silicon electrode.
According to a fourth aspect the invention comprises the use of porous silicon as a transfer medium for transferring materials into a living cell.
It has been discovered that porous silicon is biocompatible, and it has now been discovered that porous silicon can be corroded in, or resorbed into, a mammalian body without significant detrimental effect. Porous silicon can be used to locate and substantially immobilise biological material (or any substance to be introduced into a cell), with the substance being free enough once in the cell to combine with cell DNA, or otherwise be released to have an effect.
It is known from PCT Patent Application No. WO 96/10630 to have an array of micromachined bulk silicon barbs or tips and to use them mechanically to pierce the plasma membrane of large numbers of cells simultaneously. This is more efficient than piercing a single cell with a single needle, which can result in a laborious operation if hundreds of cells need to have material introduced into them. The tips of WO 96/10630 are, with hindsight, less effective at transferring material (e.g. DNA) into a pierced cell than they might be. It is, for example, proposed in that document to use surface tension forces between closely-spaced tips to hold biological material to be introduced into the cells in the spaces between the tips, and to trap it between the tips (probes) and the substrate.
A proposal is discussed in U.S. Pat. No. 5,262,128 which was published in 1993 and purports to teach the man skilled in the art to make an array of silicon needles using the Liga Processes. It is believed that this document is non-enabling at its filing (and publication) date and is not prejudicial to the novelty of the present invention for that reason. In 1989 when the application was filed, and in 1993 when it was published, the skilled man of ordinary expertise could not make very thin silicon needles having a central lumen as discussed in the document using the techniques discussed. The Liga Process is not suitable for manufacturing hollow needles in silicon, and does not enable sloping structures to be made.
U.S. Pat. No. 5,457,041 discloses an array of solid needles made of silicon having ragged tips.
U.S. Pat. No. 5,591,139 discloses a silicon microneedle that is formed in the plane of a silicon wafer.
WO 97/06101 discloses a method for producing bioactive silicon as a wafer, and suggest uses for bioactive silicon in the fabrication of biosensors and in bioassays.
WO 92/01802 discloses the idea of getting substance into a cell by incorporating the substance in a liquid and making the particles of the liquid, and then accelerating the ice particles to penetrate the cells, the ice particles melting after cell-penetration.
JP 06 034 361 discloses a porous silicon atomic force microscope tip. The device does not penetrate the surface being imaged.
U.S. Pat. No. 4,969,468 discusses solid metal needles for electrical contact with nerves.
According to another aspect the invention comprises a cell-penetrating member or micropiercer made of porous silicon.
The cell-penetrating member is adapted to have a substance to be introduced into a cell carried by the porous silicon.
According to another aspect, the invention comprises a cell penetrating member or a micropiercer comprising at least a region of porous silicon. Preferably the substance comprises DNA or RNA, a fragment of DNA or RNA, or a construct of DNA or RNA.
The cell penetrating member or micropiercer is preferably adapted to have a substance to be introduced to a cell carried by porous silicon.
The porous silicon region is adapted to immobilise a substance (e.g. DNA) in comparison with its mobility when provided with a bioinert substance such as titanium. The porous silicon region is preferably at the tip of the cell penetrating member or micropiercer. The cell penetrating member or micropiercer may be a tip or barb, with no central lumen, or it may be a needle with a central channel. The cell penetrating member or micropiercer may have a capillary or pore network extending from a reservoir or channel to a substance delivery region provided on the surface of the cell penetrating member or micropiercer.
The cell penetrating member or micropiercer may have a coating of porous silicon, or it may be porous throughout its cross-section, at least at its tip (or other substance delivery region if that is not the tip). Substantially the whole exterior surface of the cell penetrating member or micropiercer that penetrates a cell in use may comprise porous silicon.
The cell penetrating member or micropiercer may be a bulk silicon microtip with a porous silicon coating.
An advantage of holding the substance to be introduced to the cell at the tip of the cell penetrating member or micropiercer itself, instead of in channel/spaces between tips, is that the material is definitely introduced into the cell, and typically deeply into the cell. This may increase the success rate of the operation (in many cases introducing DNA into cells and stable uptake of the DNA/fragment is not statistically very successful—a few percent may succeed, which is why so many cells have to be injected).
Instead of using porous silicon to immobilise the material on the tip/ensure at least some
Beisner William H.
Nixon & Vanderhye P.C.
PSIMEDICA Limited
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