Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Fungus
Reexamination Certificate
1999-01-14
2001-06-19
Naff, David M. (Department: 1651)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Fungus
C424S408000, C428S402200, C435S177000, C435S178000, C435S182000, C435S382000
Reexamination Certificate
active
06248321
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a method for the cellular-scale encapsulation of microparticles, and to microparticles encapsulated according to the method.
BACKGROUND OF THE INVENTION
Recently, there has been considerable interest in encapsulation/immobilization of microparticles, including living cells, propagules of living cells, bacteria, viruses, fungi and the like.
Moreover, it is often desirable to formulate biological control agents so that their propagules are encapsulated and provided with water and nutrients necessary for growth. In this context, the necessity to overcome dew period requirements of weed pathogens is critical. Encapsulation can protect propagules from loss of viability due to desiccation, damage by UV light, and other environmental stresses. It can also aid in packaging inoculum in a form that is easier to manipulate and harder for pests to detect.
Simple and sometimes effective anti-desiccant formulations have spanned the range from oils to polysaccharide gets and guns, etc. There are a raft of papers on the subject, please see Auld & Morin, 1995
1
and Green et al. (1998)
2
for comprehensive overviews. There are various polymer encapsulation methods that have been derived for bacteria and other small targets. In biological control research, most attempts to encapsulate spores and other cells have involved their inclusion in various kinds of macroscopic polymer granules (alginates, pastas), e.g. see Connick et al., 1991
3
. In biological control of weeds, invert emulsions are used with increasing frequency, because they can overcome the requirement for a dew period, e.g. see Connick et al., 1991
4
.
The above methods all have limitations. Simple antidessicant gels, etc. have had limited success owing to the high viscosity needed to achieve adequate water-holding properties. In other words, such formulations cannot be easily sprayed, and require far more material than is economically feasible to apply. Invert emulsions suffer similar problems, in that they are bulky, and costly in terms of the amount of material that must be applied. They can cause collateral damage because they can be phytotoxic in their own right, and they require special equipment for application due to their high viscosity. In addition, invert emulsions must be prepared shortly before application, and do not allow for freeze-drying or other types of long term storage. Regarding dry formulations, macroscopic granules containing eukaryotic cells cannot be reduced to a microscopic size without crushing and killing cells. This is unfortunate, because macroscopic granules cannot be easily sprayed and they do not efficiently distribute inoculum or other materials because of their relatively low surface area to volume ratio. Vapor coating methods are also impractical in our experience, due to the cost of the method, the cumbersome equipment required, the exposure of cells to relatively high heat during some coating methods, the dry, piecemeal nature of coatings produced at lower heats, and the larger size of the particles produced. A method is needed to make formulations a more intrinsic part of cells, hence encapsulation.
Currently applicable encapsulation or immobilization techniques tend to produce polyacrylamide or alginate beads in the range of 0.5-1 mm in diameter, too large to be of practical use for cellular-scale applications. They tend to be expensive or unwieldy and rely on methods, equipment, or chemicals that are fairly specialized. It is possible to place aqueous droplets containing acrylamides or other monomers into dispersion media composed of hydrophobic solvents or oils, wherein the drops are held in place as spheres while the monomers polymerize. This method comes closest to our approach, e.g. see Nilsson et al., 1983
5
, also see U.S. Pat. No. 4,647,536, but the technique is unwieldy, mostly because the polymerization is proceeding as the drops are forming globules in the solvent.
A major advantage of the present methodology is that we avoid the complicated prior art procedures to extract the capsules from dispersion media, i.e. the capsules form on their way out of the dispersion medium, killing two birds with one stone. This is not only a unique method, but also results in a unique characteristic—the capsules are not encumbered by any significant (visible in the compound microscope) oil coating. There are occasionally some small oil droplets that can be seen in and outside the capsules, but these are a very small portion of the overall material that is produced. Freeing the capsules from the dispersion medium in this manner is desirable for a number of reasons. First, materials with an oily consistency may be difficult to manipulate, concentrate, or formulate for practical applications. Second, the presence of significant amounts of the dispersion medium may affect the performance or behavior of the encapsulated material. Third, the presence of an extra component could complicate legal registration or other types of regulatory approval necessary for commercialization of products. Fourth, on larger scales, aqueous solutions are cheaper than oil solutions. There is another advantage to polymerizing upon exit from the dispersion medium: while Nilsson's stirring method avoids adhesion or bonding (perhaps even fusion) of capsules polymerizing inside the dispersion medium, our method does not require such fine control over agitation, and is thus more reliable and easier to reproduce, particularly if the method is to be scaled-up in volume.
SUMMARY OF THE INVENTION
It is an object of the present invention to encapsulate microparticles e.g. propagules of eukaryotic biocontrol agents with cellular-scale polymer capsules, via a novel, inexpensive method that encapsulates with as much as 100% efficiency.
According to one aspect of the invention, a method is provided for cellular-scale encapsulation of microparticles, comprising:
(i) providing an aqueous suspension of the microparticles and a polymer precursor, the polymer being biodegradable and not requiring heat or pressure to form,
(ii) adding a non-cytotoxic, hydrophobic dispersion medium containing an emulsifier, to form an aqueous dispersion,
(iii) agitating vigorously to form a stable emulsion of microscopic globules including a microparticle and the polymer precursor, and
(iv) adding the suspension to an aqueous solution containing a polymerization agent/catalyst, to polymerize and precipitate the globules and form polymer encapsulated microparticles of cellular scale dimensions.
REFERENCES:
patent: 4352883 (1982-10-01), Lim
patent: 4409331 (1983-10-01), Lim
patent: 4647536 (1987-03-01), Mosbach et al.
patent: 4724147 (1988-02-01), Marois et al.
patent: 5015576 (1991-05-01), Nilsson et al.
patent: 5786188 (1998-07-01), Lamar et al.
patent: 5879712 (1999-03-01), Bomberger et al.
Wheeler Jeffery Jerome
Winder Richard Scott
Anderson J. Wayne
Her Majesty the Queen in right of Canada as represented by the
Naff David M.
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